Engineered red blood cells having rare antigen phenotypes

ABSTRACT

Provided herein are engineered red blood cells expressing rare blood antigen group profiles, and methods of making use the same, are described. Also provided are recombinant reagent red blood cells that express or lack the expression of at least one protein (e.g., a blood group antigen) on its surface and uses thereof.

PRIORITY CLAIM

This application is a national phase application under 35 U.S.C, § 371of International Application No. PCT/US2018/057932, filed Oct. 29, 2018,which claims benefit of priority to U.S. Provisional Patent ApplicationNo. 62/578,263, filed Oct. 27, 2017, and U.S. Provisional PatentApplication No. 62/578,768, filed Oct. 30, 2017, the entire contents ofeach application being hereby incorporated by reference.

Pursuant to 37 C.F.R. 1.821(c), a sequence listing is submitted herewithas an ASCII compliant text file named “CHOPP0014US_ST25.txt”, created onApr. 20, 2020 and having a size of ˜1 kilobytes. The content of theaforementioned file is hereby incorporated by reference in its entirety.

GOVERNMENT SUPPORT

This invention was made with government support under grant nos.HL130764-01 and HL134696-01 awarded by the National Institutes ofHealth. The government has certain rights in the invention.

I. TECHNICAL FIELD

The present disclosure relates to the fields of molecular biology,medicine and immunology, More specifically, it relates to engineered redblood cells having rare antigen phenotypes and/or the production ofrecombinant reagent red blood cells that do not express at least oneantigen on its surface or express rare or unique combinations ofantigens not found in natural human populations and uses of the same,e.g., their use in testing for blood type antibody reactivity intransfusion settings.

II. BACKGROUND

Transfusion of red blood cells (RBCs) is routinely used for manyclinical and surgical applications. RBC transfusions were developed over80 years ago, before all other cellular therapies, and are one of themost frequently used life-saving medical procedures. Blood transfusionsare also a primary treatment for blood loss and hereditary anemia,including ϑ-thalassemia major and sickle cell disease (SCD). Accordingto the American Association of Blood Banks, 29 million units of bloodcomponents are transfused annually in the United States. This procedurehas saved many lives. The demand for such transfusions continues toincrease with advances in medical treatments and an aging population.

There are more than 30,000 patients transfused at least 6 times a yearin the U.S. for chronic anemia and many are allo-immunized. Antibodyscreening against commercial reagent cells is performed prior to eachtransfusion. The current estimated cost for antibody identification inallo-immunized patients prior to transfusion is $100 to $200 million peryear in the United States. Referral laboratory testing and performingmultiple adsorptions to detect underlying alloantibodies contributessignificantly ($500 to $2,000/per event) to this cost. Development ofnovel reagent red cells could reduce these costs by approximately 70%while improving safety.

Unfortunately, a major challenge is the high rate of alloimmunization(antibody formation against transfused red blood cells) that occurs intransfused patients. Genetic diversity in blood group antigens inpatients of African descent (relevant for treatment of sickle celldisease), as compare to the primarily European-based blood donor pool,contributes to this high incidence and complexity of antibodies found inpatients with sickle cell disease. At present, there is no readysolution to this challenging problem.

In order to better manage transfusion alloimmune responses, it would bemost useful to be able to easily screen patients for alloimmunity, withan eye towards using such information to avoid use of blood productsmore likely to trigger dangerous reactions. In particular, a panel ofrare blood types that would permit rapid screening.

SUMMARY

A major problem for transfusion therapy for chronic anemia is the highdegree of genetic diversity in blood group antigens in people of Asianand African backgrounds compared to white Europeans, who are themajority of blood donors. These polymorphic antigens on RBCs contributeto the high incidence of allo-immunization and presence of multipleantibodies in the serum of these patients. Identifying the specificityof these antibodies and determining if they are auto- or allo-antibodiesis critical for providing compatible blood or blood products. However,the current process can be complex, often involves costly referencelaboratory testing, lacks standardization, and is hampered by the lackof suitable reagent RBCs for timely provision of blood products.

Recombinant reagent cultured red blood cells (cRBCs) will facilitateantibody identification in allo-immunized multiply transfused patients,and streamline and standardize testing.

The present disclosure is based, at least in part, on the generation ofa novel and improved system for displaying recombinant proteins on thesurface of eukaryotic cells (e.g., mammalian cells, e.g., reagent redblood cells). As elaborated on below and exemplified in the workingexamples, the system offers several benefits over those previouslydescribed.

Thus, in accordance with the present disclosure, there is provided aplurality of antigenically distinct engineered red blood cells (RBCs),wherein said plurality of RBCs exhibit distinct blood antigen groupprofiles, including at least two rare blood antigen groups. Theplurality of RBCs may exhibit at least three, four, five, six, seven,eight, nine, ten or fifteen distinct blood antigen groups. The pluralityof RBCs may be immortalized from naturally-occurring isolated RBCs, suchas by transfecting a naturally-occurring RBC with a construct expressinga transforming oncoprotein. The plurality of RBCs may be produced frominduced pluripotent stem cells, such as those produced from inducedpluripotent stem cells using CRISPR to insert, delete or disrupt acoding sequence for one or more blood antigens.

The plurality of RBCs may comprise two or more of the following bloodantigen group profiles: Rh null, D −/−, U/S/s antigen negative(glycophorin B null), RHCE negative, positive for low prevalence RhDantigens, Dombrok (Do) null, and Lutheran null. The plurality of RBCsmay further comprise one or more of Kell positive, Kidd positive, Duffypositive and MNS antigen positive. The plurality of RBCs may comprisethree, four, five or all six blood antigen group profiles.

In another embodiment there is provided a plurality of antigenicallydistinct induced pluripotent stem cells (IPSCs), wherein said pluralityof IPSCs exhibit distinct blood antigen group profiles, including atleast two rare blood antigen groups. The plurality of IPSCs may exhibitat least three, four, five, six, seven, eight, nine, ten or fifteendistinct blood antigen groups. The plurality of IPSCs may be producedfrom induced pluripotent stem cells using CRISPR to insert, delete ordisrupt a coding sequence for one or more blood antigens. The pluralityof IPSCs may comprise two or more of the following blood antigen groupprofiles: Rh null, D −/−, U/S/s antigen negative (glycophorin B null),RHCE negative, positive for low prevalence RhD antigens, Dombrok (Do)null, and Lutheran null. The plurality of IPSCs may further comprise oneor more of Kell positive, Kidd positive, Duffy positive and MNS antigenpositive. The IPSCs may comprise three, four, five or all six bloodantigen group profiles.

In yet another embodiment, there is provided a plurality ofantigenically distinct engineered erythroblasts, wherein said pluralityof erythroblasts exhibit distinct blood antigen group profiles,including at least two rare blood antigen groups. The plurality oferythroblasts may exhibit at least three, four, five, six, seven, eight,nine, ten or fifteen distinct blood antigen groups. The plurality oferythroblasts may be produced from induced pluripotent stem cells. Theplurality of erythroblastis may comprise two or more of the followingblood antigen group profiles: Rh null, D −/−, U/S/s antigen negative(glycophorin B null), RHCE negative, positive for low prevalence RhDantigens, Dombrok (Do) null, and Lutheran null, and optionally furthercomprise one or more of Kell positive, Kidd positive, Duffy positive andMNS antigen positive. The plurality of erythroblasts may comprise three,four, five or all six blood antigen group profiles.

In still yet another embodiment, there is provided a method ofidentifying blood antigen group-specific antibodies in a subjectcomprising (a) obtaining a subject sample comprising antibodies; (b)contacting said sample with the plurality of antigenically distinctengineered RBCs as described above; and (c) identifying binding of saidantibodies to one or more of said antigenically distinct engineeredRBCs. Step (c) may comprise an agglutination assay, such as a gel cardassay, or step (c) may comprise an ELISA, an RIA or flow cytometry.

The subject may be a human subject. The subject may suffer from adisease treating by blood transfusion, such as sickle cell disease,thalassemia, a hemoglobinopathy, congenital anemias (e.g., pyruvatekinase deficiency; blackfan anemia), bone marrow failure syndromes,myelodysplastic syndromes, multiple myeloma, or cancer. The sample maybe whole blood, plasma, or serum. The subject may have previously had ablood transfusion or a pregnancy. The subject may not have previouslyhad a blood transfusion or a pregnancy.

In still a further embodiment, there is provided a method of producingan engineered red blood cell (RBC) comprising (a) providing an inducedpluripotent stem cell (iPSC); (b) modifying said iPSC to express a bloodgroup antigen and/or not express a blood group antigen; and (c) inducingdifferentiation of said iPSC into an erythroblast and thereafter into amature RBC. The method may further comprise causing said RBC to becomeenucleated. The RBC may be Rh null, D −/−, U/S/s antigen negative(glycophorin B null), RHCE negative, positive for low prevalence RhDantigens, Dombrok (Do) null, or Lutheran null. Modifying may compriseuse of CRISPR to insert, delete or disrupt a coding sequence for one ormore blood antigens.

Provided herein are recombinant red blood cells, wherein the recombinantred blood cells have a surface phenotype selected from the groupconsisting of: (i) D⁻, C⁻, E⁻, c⁻, e⁻; (ii) D⁺, C⁻, E⁻, c⁻, e⁻; (iii)D⁻, U⁻, S⁻, s⁻; (iv) D⁻, hrB⁻, VS⁺; (v) D⁻, hrB⁻, hrS⁻; (vi) C⁻, E⁻, c⁻,e⁻; (vii) D⁺, C⁻, E⁻, c⁻, e⁻, Go(a)⁺; (viii) D⁺, C⁻, E⁻, c⁻, e⁻, DAK⁺;(ix) D⁻, Doa⁻, Dob⁻; (x) Lua⁻ b⁻; (xi) CD47⁻; and (xii) any combinationof two or more of the surface phenotypes of (i) to (xi).

Provided herein are recombinant red blood cells, wherein the recombinantred blood cells are characterized by the absence of at least one or morecell surface antigens on its surface selected from the group consistingof: a C antigen, an E antigen, a c antigen, an e antigen, a U antigen,an S antigen, an s antigen, an hrB antigen, a Lua antigen, a Lub antigenand a CD47 antigen.

In some embodiments, the recombinant red blood cell is characterized bythe absence of at least ten of the one or more cell surface antigens.

In some embodiments, the recombinant red blood cell is characterized bythe absence of at least eight of the one or more cell surface antigens.

In some embodiments, the recombinant red blood cell is characterized bythe absence of at least four of the one or more cell surface antigens.

In some embodiments, the recombinant red blood cell is characterized bythe absence of at least two of the one or more cell surface antigens.

In some embodiments of any of the recombinant red blood cells describedherein, the recombinant red blood cell is further characterized by theabsence of a D antigen on its cell surface.

In some embodiments of any of the recombinant red blood cells describedherein, the recombinant red blood cell is further characterized by thepresence of a D antigen on its cell surface.

In some embodiments of any of the recombinant red blood cells describedherein, the recombinant red blood cell is further characterized by thepresence of a Go(a) antigen on its cell surface.

In some embodiments of any of the recombinant red blood cells describedherein, the recombinant red blood cell is further characterized by thepresence of a DAK antigen on its cell surface.

In some embodiments of any of the recombinant red blood cells describedherein, the recombinant red blood cell is characterized by the absenceof a Doa antigen and a Dob antigen on its cell surface.

Also provided herein are kits that include: a solid support; a firstreagent red blood cell characterized by the presence of one or more cellsurface antigens on its surface; and a second reagent red blood cellcharacterized by the absence of at least one of the one or more cellsurface antigens on its surface; wherein: the one or more cell surfaceantigens are selected from the group consisting of: a C antigen, an Eantigen, a c antigen, an e antigen, a U antigen, an S antigen, an santigen, an hrB antigen, a Lua antigen, a Lub antigen and a CD47antigen; and the first reagent red blood cell and the second reagent redblood cell have the same surface phenotype, except for the at least onecell surface antigen.

In some embodiments of any of the kits described herein, the kit furtherincludes one or more additional reagent red blood cell, wherein: eachadditional reagent red blood cell has a surface phenotype that ischaracterized, at least in part, by the absence of one or more cellsurface antigens; and each reagent red blood cell present in the kit hasa different surface phenotype as compared to all the other reagent redblood cells in the kit.

In some embodiments, the kit includes at least 10 additional reagent redblood cells.

In some embodiments, the second reagent red blood cell is characterizedby the absence of a C antigen, an E antigen, a c antigen, and an eantigen on its cell surface.

In some embodiments, the second reagent red blood cell is furthercharacterized by the absence of a D antigen on its cell surface.

In some embodiments, the second reagent red blood cell is furthercharacterized by the presence of a D antigen on its cell surface.

In some embodiments, the second reagent red blood cell is characterizedby the absence of a U antigen, a S antigen, and a s antigen on its cellsurface.

In some embodiments, the second reagent red blood cell is furthercharacterized by the absence of a D antigen on its cell surface.

In some embodiments, the second reagent red blood cell is characterizedby the presence of a D antigen on its cell surface and the absence of aC antigen, an E antigen, a c antigen, and an e antigen on its cellsurface.

In some embodiments, the second reagent red blood cell is furthercharacterized by the presence of a Go(a) antigen on its cell surface.

In some embodiments, the second reagent red blood cell is furthercharacterized by the presence of a DAK antigen on its cell surface.

In some embodiments, the second reagent red blood cell is characterizedby the absence of a Doa antigen and a Dob antigen on its cell surface.

In some embodiments, the second reagent red blood cell is characterizedby the absence of an hrB antigen on its cell surface.

In some embodiments, the second reagent red blood cell is furthercharacterized by the absence an hrS antigen on its cell surface.

In some embodiments, the second reagent red blood cell is furthercharacterized by the absence a D antigen on its cell surface.

In some embodiments, the second reagent red blood cell is furthercharacterized by the presence of a VS antigen on its cell surface.

In some embodiments, the second reagent red blood cell is characterizedby the absence of a C antigen, an E antigen, a c antigen, an e antigen,a U antigen, a S antigen, an s antigen and an hrB antigen on its cellsurface.

In some embodiments, the second reagent red blood cell is characterizedby a phenotype selected from the group consisting of: (i) D⁻, C⁻, E⁻,c⁻, e⁻; (ii) D⁺, C⁻, E⁻, c⁻, e⁻; (iii) D⁻, U⁻, S⁻, s⁻; (iv) D⁻, hrB⁻,VS⁺; (v) D⁻, hrB⁻, hrS⁻; (vi) C⁻, E⁻, c⁻, e⁻; (vii) D⁺, C⁻, E⁻, c⁻, e⁻,Go(a)⁺; (viii) D⁺, C⁻, E⁻, c⁻, e⁻, DAK⁺; (ix) D⁻, Doa⁻, Dob⁻; (x)Lua⁻b⁻; (xi) CD47⁻; and (xii) any combination of two or more of thesurface phenotypes of (i) to (xi).

In some embodiments, the second reagent red blood cell is characterizedby the absence of a C antigen, an E antigen, a c antigen, an e antigen,a U antigen, a S antigen, an s antigen and an hrB antigen on its cellsurface.

In some embodiments of any of the kits described herein, the solidsupport is selected from the group consisting of: a gel card, amulti-well assay plate, an array, a microplate, a film, a tube, a well,a capillary, a paper matrix, a slide, and a chip.

Provided herein are methods of determining blood group antigencompatibility of a patient sample that include: (a) contacting a firstreagent red blood cell with a patient sample containing a plurality ofantibodies; wherein the first reagent red blood cell is characterized bythe presence of one or more blood group antigens on its surface; and (b)contacting a second reagent red blood cell with the patient sample;wherein the second reagent red blood cell is characterized by theabsence of at least one of the one or more cell surface antigens on itssurface; (c) detecting whether agglutination occurs upon contacting thefirst reagent red blood cell with the patient sample; (d) detectingwhether agglutination occurs when contacting the second reagent bloodcell with the patient sample; and (e) identifying that the patientsample is compatible with the at least one of the one or more cellsurface antigens when no agglutination is detected in steps (c) and (d),or identifying that the patient sample is not compatible with the atleast one of the one or more cell surface antigens when agglutination isdetected in step (c) but is not detected in step (d), wherein: the oneor more cell surface antigens are selected from the group consisting of:a C antigen, an E antigen, a c antigen, an e antigen, a U antigen, an Santigen, an s antigen, and an hrB antigen, and the first reagent redblood cell and the second reagent red blood cell have the same surfacephenotype, except for the at least one cell surface antigen.

In some embodiments, identifying that the patient sample is notcompatible with the at least one of the one or more cell surfaceantigens when agglutination is detected in step (c) but is not detectedin step (d).

In some embodiments, the method includes the use of one or moreadditional reagent red blood cells, wherein: each additional reagent redblood cell has a surface phenotype that is characterized, at least inpart, by the absence of one or more cell surface antigens; and eachreagent red blood cell used in the method has a different surfacephenotype as compared to all the other reagent red blood cells used inthe method.

In some embodiments, the method includes the use of ten or moreadditional reagent red blood cells.

In some embodiments, the first reagent red blood cell is characterizedby the presence of a C antigen, an E antigen, a c antigen, and an eantigen on its cell surface.

In some embodiments, the first reagent red blood cell is furthercharacterized by the presence of a D antigen on its cell surface.

In some embodiments, the first reagent red blood cell is furthercharacterized by the absence of a D antigen on its cell surface.

In some embodiments, the second reagent red blood cell is characterizedby the absence of a U antigen, an S antigen, and an s antigen on itscell surface.

In some embodiments, the second reagent red blood cell is furthercharacterized by the absence of a D antigen on its cell surface.

In some embodiments, the second reagent red blood cell is characterizedby the absence of a D antigen on its cell surface and the absence of a Cantigen, an E antigen, a c antigen, and an e antigen on its cellsurface.

In some embodiments, the second reagent red blood cell is furthercharacterized by the presence of a Go(a) antigen on its cell surface.

In some embodiments, the second reagent red blood cell is furthercharacterized by the presence of a DAK antigen on its cell surface.

In some embodiments, the second reagent red blood cell is characterizedby the presence of a Doa antigen and a Dob antigen on its cell surface.

In some embodiments, the second reagent red blood cell is characterizedby the absence of an hrB antigen on its cell surface.

In some embodiments, the second reagent red blood cell is furthercharacterized by the absence an hrS antigen on its cell surface.

In some embodiments, the second reagent red blood cell is furthercharacterized by the absence a D antigen on its cell surface.

In some embodiments, the second reagent red blood cell is furthercharacterized by the presence of a VS antigen on its cell surface.

In some embodiments, the second reagent red blood cell is characterizedby the absence of a C antigen, an E antigen, a c antigen, an e antigen,a U antigen, an S antigen, an s antigen, a hrS antigen and a hrB antigenon its cell surface.

In some embodiments, the second target red blood cell is characterizedby a phenotype selected from the group consisting of: (i) D⁻, C⁻, E⁻,c⁻, e⁻; (ii) D⁺, C⁻, E⁻, c⁻, e⁻; (iii) D⁻, U⁻, S⁻, s⁻; (iv) D⁻, hrB⁻,VS⁺; (v) D⁻, hrB⁻, hrS⁻; (vi) C⁻, E⁻, c⁻, e⁻; (vii) D⁺, C⁻, E⁻, c⁻, e⁻,Go(a)⁺; (viii) D⁺, C⁻, E⁻, c⁻, e⁻, DAK⁺; (ix) D⁻, Doa⁻, Dob⁻; and (x)any combination of two or more of the surface phenotypes of (i) to (ix).

In some embodiments of any of the methods described herein, the methodfurther includes: selecting a tissue or blood product that is compatiblewith the at least one of the one or more cell surface antigens for apatient having its patient sample identified as being compatible withthe at least one of the one or more cell surface antigens; or selectinga tissue or blood product that does not include the at least one of theone or more cell surface antigens for a patient having its patientsample identified as not being compatible with the at least one of theone or more cell surface antigens.

In some embodiments, the method further includes: administering theselected tissue or blood product that is compatible with the at leastone of the one or more cell surface antigens to the patient having itspatient sample identified as being compatible with the at least one ofthe one or more cell surface antigens; or administering the selectedtissue or blood product that does not include the at least one of theone or more cell surface antigens to the patient having its patientsample identified as not being compatible with the at least one of theone or more cell surface antigens.

In some embodiments, the patient has hereditary anemia. In someembodiments, the patient has β-thalassemia. In some embodiments, thepatient has sickle cell disease.

Also provided herein are methods of determining blood compatibility of apatient sample that include: (a) contacting a first reagent red bloodcell with a patient sample containing a plurality of antibodies; whereinthe first reagent red blood cell is characterized by the presence of oneor more cell surface antigens; and (b) contacting a second reagent redblood cell with the patient sample; wherein the second reagent red bloodcell is characterized by the absence of at least one of the one or morecell surface antigens on its surface; (c) detecting whetheragglutination occurs upon contacting the first reagent red blood cellwith the patient sample; (d) detecting whether agglutination occurs whencontacting the second reagent blood cell with the patient sample; and(e) identifying that the patient sample is compatible with the at leastone of the one or more cell surface antigens when no agglutination isdetected in steps (c) and (d), or identifying that the patient sample isnot compatible with the at least one of the one or more cell surfaceantigens when agglutination is detected in step (c) but is not detectedin step (d), wherein: the one or more cell surface antigens are selectedfrom the group consisting of: a Lua antigen, a Lub antigen, and a CD74antigen, and the first reagent red blood cell and the second reagent redblood cell have the same surface phenotype, except for the at least onecell surface antigen.

In some embodiments, the method includes the use of one or moreadditional reagent red blood cells, wherein: each additional reagent redblood cell has a surface phenotype that is characterized, at least inpart, by the absence of one or more cell surface antigens; and eachreagent red blood cell used in the method has a different surfacephenotype as compared to all the other reagent red blood cells used inthe method.

In some embodiments, the method includes the use of ten or moreadditional reagent red blood cells.

In some embodiments, the second reagent red blood cell is characterizedby the absence of a Lua antigen and a Lub antigen on its cell surface.

In some embodiments, the second reagent red blood cell is characterizedby the absence of a CD47 antigen.

In some embodiments, the sample is obtained from a patient that hasreceived an anti-CD38 immunotherapy.

In some embodiments, the sample is obtained from a patient that hasreceived an anti-CD47 immunotherapy.

In some embodiments of any of the methods described herein, the methodfurther includes: selecting a tissue or blood product that is compatiblewith the at least one of the one or more cell surface antigens for apatient having its patient sample identified as being compatible withthe at least one of the one or more cell surface antigens; or selectinga tissue or blood product that does not include the at least one of theone or more cell surface antigens for a patient having its patientsample identified as not being compatible with the at least one of theone or more cell surface antigens.

In some embodiments, the method further includes: administering theselected tissue or blood product that is compatible with the at leastone of the one or more cell surface antigens to the patient having itspatient sample identified as being compatible with the at least one ofthe one or more cell surface antigens; or administering the selectedtissue or blood product that does not include the at least one of theone or more cell surface antigens to the patient having its patientsample identified as not being compatible with the at least one of theone or more cell surface antigens.

In some embodiments of any of the methods described herein, the patientsample is a plasma sample or a serum sample.

In some embodiments of any of the methods described herein, the methodfurther includes: transfusing a therapeutically effective amount of asecond reagent red blood cell to the patient having its patient sampleidentified as being compatible with the at least one of the one or morecell surface antigens; and, wherein the second reagent red blood cellhas a phenotype selected from the group consisting of: (i) Lua⁻ b⁻; and(ii) CD47⁻.

As used herein, the terms “reagent red blood cell,” “reagent culturedred blood cell” and “recombinant red blood cell” are usedinterchangeably. A reagent red blood cell has all of the functional andmorphological characteristics of a naturally-occurring red blood, buthas been genetically modified (or a precursor cell to the reagent redblood cell has been genetically modified) in vitro. In some embodiments,reagent red blood cells described herein can lack the expression of oneor more blood group antigens that are expressed on naturally-occurringred blood cells, or express rare antigens or combinations of antigensnot found often, if ever, in humans.

The term “surface phenotype” refers to the collection of antigenspresent on the surface of a red blood cell. In some embodiments, anantigen present on the surface of a red blood cell can be atransmembrane protein or a carbohydrate moiety on a protein. In someembodiments, an antigen present on the surface of a red blood cell canbe covalently attached to the plasma membrane of a red blood cell (e.g.,GPI-anchored). In some embodiments, an antigen present on the surface ofa red blood cell can be non-covalently bonded to the plasma membrane ofa red blood cell (e.g., non-covalently bonded to a transmembrane proteinin the plasma membrane of the red blood cell). For example, a firstreagent red blood cell and a second reagent red blood can have the sameor an indistinguishable surface phenotype except for the presence orabsence of at least one cell surface antigen.

As used herein an array can, in some embodiments, refer to an orderedarrangement of bound antibodies that specifically bind to red blood cellantigens (e.g., any of the red blood cell antigens described herein) ona support surface (e.g., a microarray, a chip, a slide, a film, a goldcoated surface, tubing, polymers, microparticles, plates, tubing,magnetic or nonmagnetic beads). In some aspects an array include atleast about 2 antibodies (e.g., at least about 4 antibodies, at leastabout 6 antibodies, at least about 8 antibodies, at least about 10antibodies). In some aspects, an array is made of glass, silicon,silicon oxide, metal oxides, metal, polymers (e.g., poly-L-lysine,aminopropylsilane, carboxysilane), hydrogels and polymer-brushes. Insome aspects, the array is planar or spheroid. In some aspects, an arrayis about 20 mm to about 200 mm (e.g., about 20 mm to about 180 mm, about20 mm to about 160 mm, about 20 mm to about 140 mm, about 20 mm to about120 mm, about 20 mm to about 100 mm, about 20 mm to about 80 mm, about20 mm to about 60 mm, about 20 mm to about 40 mm, about 30 mm to about200 mm, about 30 mm to about 180 mm, about 30 mm to about 160 mm, about30 mm to about 140 mm, about 30 mm to about 120 mm, about 30 mm to about100 mm, about 30 mm to about 80 mm, about 30 mm to about 60 mm, about 40mm to about 200 mm, about 40 mm to about 180 mm, about 40 mm to about180 mm, about 40 mm to about 160 mm, about 40 mm to about 140 mm, about40 mm to about 120 mm, about 40 mm to about 100 mm, about 40 mm to about80 mm, about 40 mm to about 60 mm, or about 100 mm to about 200 mm),with up to 10,000 spots of antibodies (e.g., about 2 spots to about10,000 spots, about 2 spots to about 2,000 spots, about 2 spots to about1,000 spots, and any range in between). An array can be used to testmore than one sample (e.g., 2, 4, 10, 20, 40, 60, 80 or 100 samples).

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Methods and materials aredescribed herein for use in the present invention; other, suitablemethods and materials known in the art can also be used. The materials,methods, and examples are illustrative only and not intended to belimiting. All publications, patent applications, patents, sequences,database entries, and other references mentioned herein are incorporatedby reference in their entirety. In case of conflict, the presentspecification, including definitions, will control.

It is contemplated that any method or composition described herein canbe implemented with respect to any other method or composition describedherein.

Other objects, features and advantages of the present disclosure willbecome apparent from the following detailed description and figures, andfrom the claims. It should be understood, however, that the detaileddescription and the specific examples, while indicating specificembodiments of the disclosure, are given by way of illustration only,since various changes and modifications within the spirit and scope ofthe disclosure will become apparent to those skilled in the art fromthis detailed description.

DESCRIPTION OF THE DRAWINGS

The following drawings form part of the present specification and areincluded to further demonstrate certain aspects of the presentdisclosure. The disclosure may be better understood by reference to oneor more of these drawings in combination with the detailed descriptionof specific embodiments presented herein.

FIG. 1 . RBC panel for antibody detection. Each row represents the RBCantigen profile of a single donor and columns show presence (+) orabsence (0) of the indicated antigen. Reagents 1-3 are standardcommercial RBCs used for antibody detection. Autocontrol=patient RBCswhich include endogenous and transfused RBCs. Bottom 3 rows representrare reagent RBCs. Far right column shows graded reactivity of patient'sserum to each reagent RBC.

FIG. 2 . RBC agglutination grading. Agglutination occurs when antibodypresent reacts against an RBC antigen, and is graded 0 to 4+. The gelassay depicted shows agglutinated RBCs retained in the column matrix,and free nonagglutinated cells pellet at the bottom.

FIGS. 3A-D. Alloimmunization in 182 patients with SCD transfused with D,C, E, K-matched RBCs from minority donors. FIG. 3A. Blue tag thataccompanies RBC donation from a self-identified African American donor.FIG. 3B. 146 antibodies were identified and 70% had Rh specificity. FIG.3C. 56 Rh antibodies occurred in patients whose RBCs typed positive forthat antigen (sometimes mistaken for “autoantibodies”), and 35 wereidentified in antigen-negative patients despite transfusion withRh-matched RBCs. FIG. 3D. % Hemoglobin S and hemoglobin ofrepresentative patient with altered RH, receiving RBC transfusions every3 weeks. Arrows indicate new anti-e detection. Grey boxes represent+/−1.5 SD the calculated mean hemoglobin S and hemoglobin.

FIG. 4 . RHD and RHCE alleles found in 857 patients with SCD and 587African American donors. Each grey box represents 1 of 10 exons in theRH genes. Black boxes represent exon exchange between RHD and RHCE.Vertical lines indicate position in the exon encoding amino acidsubstitutions in the protein. Dashed line indicates gene deletion.Arrowhead indicated 37-bp duplication. Hatched boxes represent exonsencoding a frameshift and untranslated region of the inactive RHDpseudogene.

FIGS. 5A-C. Gene-targeting with the CRISPR/Cas9 system. FIG. 5A.Schematic of targeting the ARX locus. The guide RNA of the CRISPR-Cas9system targets a DNA break near the ATG. Using a gene-targeting vector,red fluorescent protein (Myr-RFP) is inserted into the beginning of ARX.FIG. 5B. Southern Blot showing correct gene targeting FIG. 5C. TheARX-RFP reporter cell line was differentiated and ARX visualized by RFP.

FIGS. 6A-B. Generation of a FLI-1 overexpression line. FIG. 6A.Schematic of targeting strategy (PR: puromycin resistance; Gp1ba: Gp1bapromoter; Hom-L: homology arm left; Hom-R: homology arm right; ZFN: zincfinger nuclease site; con: control). H9 ESCs with and without the FLI-1overexpression construct were differentiated into hematopoietic cells.FIG. 6B. QRTPCR expression analysis of FLI-1 in purified Megs.

FIG. 7 . CRISPR/Cas9 gene editing strategy to mutate RHCE by homologousrecombination. Guide RNAs target a sequence close to the desiredmutation site. If mutant oligonucleotide sequence is introduced,restriction enzyme digestion results in two fragments. Sequences are,from top to bottom, SEQ ID NOS: 1, 2 and 3. GRCh38.p 12 accession numberis GCA_000001405.27. RefSeq assembly number is GCA_000001405.3.

FIGS. 8A-B. Characteristics of induced pluripotent stem cell derived redblood cells (iRBCs). (FIG. 8A) Cell surface expression of red cellmarkers on iRBCs compared to donor derived RBCs. (FIG. 8B) iRBC sizeshown by forward scatter (FSC) on flow cytometry histogram and bycytospin as compared to donor derived RBCs.

FIGS. 9A-C. Accurate Rh typing of induced pluripotent stem cell derivedred blood cells (iRBCs). Using monoclonal Rh typing reagents for D, C,c, E, and e, typing was performed with 500,000 iRBCs per gel card assaycolumn for Untargeted D− wild type (WT) iRBCs (FIG. 9A), untargeted D+WT iRBCs (FIG. 9B), and CRISPR-targeted Rh null iRBCs (FIG. 9C). The Rhphenotype predicted by genotype is shown in light grey boxes. iRBCsagglutinated and remained at the top of the gel matrix when the antigenwas predicted to be expressed (+) and did not agglutinate and pelletedto bottom of column when antigen was predicted to be absent (−).

FIGS. 10A-C. Definitive HPC pathway. FIG. 10A. Schematic representationbeginning with ESCs or iPSCs and ending with HPCs. Both hematopoieticprograms, Primitive (top) and Definitive (bottom), transition throughintermediates, developing from KDR+ CD34-CD144-progenitors that aredistinguished by CD235a expression. The generation of primitiveprogenitors (KDR+CD235a+) depends on stage-specific Activin/nodalsignaling and inhibition of the Wnt/β-catenin pathway, vs specificationof definitive progenitors (KDR+ CD235a−) requires Wnt/β-cateninsignaling during the same timeframe and co-culture with Notch ligandexpressing stroma. FIG. 10B. Morphology and relative size of theerythroid colonies (top) and terminally differentiated RBCs derived fromthese colonies (bottom). Gray arrows indicate small, primitive(Prim-RBC) colonies. Original magnification: colonies, X100; cells,X1000. FIG. 10C. Globin analysis was performed on red cell colonies fromPrim-HPCs, definitive (Def)-HPCs, fetal liver HPCs (FL), and bone marrowHPCs (BM) by QRT-PCR. In the graph, “epsilon” is represented by the leftbar, “gamma” is represented by the middle bar, and “beta” is representedby the right bar, for each set of three bars.

FIGS. 11A-B. IFC analysis of hESC derived RBC maturation. FIG. 11A.Maturational series of ESC cells matured towards definitive RBCs asdescribed in FIGS. 10A-C. Definitive HPCs were grown in liquid culturefor 12 days in RBC maturation media and analyzed based on cell size andnuclear size (Left). Bright field (BF), CD235, CD71, and DNA channelsare shown for cells indicated on left panel (Right). FIG. 11B. Thecultures were analyzed for RNA at the single cell level using RNA flow(Primeflow, Ebioscience) for epsilon and gamma globin. As expected, allcells are gamma positive, but a subset of ESC-derived definitivecultures are epsilon globin-positive while human fetal liver-derivedRBCs are negative.

FIG. 12 . ESCs expressing doxycycline-regulated Gata1 shRNAs. Top:targeting vector. Middle: modified Hprt locus in A2lox.cre parentalESCs. Bottom: the modified locus after cre-mediated recombination. Gata1or control shRNAs flanked by Mir 30 processing sequences are regulatedby tet response element (TRE). P1-P3 and loxin are PCR products used toassess the modified locus.

FIG. 13A-D. Dox-regulated suppression of Gata1 generates self-renewingMEPs. FIG. 13A. Modified ESCs were differentiated with dox, TPO and SCF.Cumulative cell number vs. time (n=4 experiments). FIG. 13B. FACSanalysis of Kit and CD41 expression. FIG. 13C. RT-PCR quantification ofGata1 mRNA after dox withdrawal. (n=3 expts)*, p<0.05. Mean±S.E.M. FIG.13D. Western blotting for GATA1 protein after dox removal.

FIG. 14 . G1ME2 cell colonies. Cells (10⁴) were plated intomethycellulose colony forming assays with EPO, SCF, TPO, IL-3, andM-CSF, in the absence of dox. Erythroid (Ery), mixederythroidmegakaryocyte (E-meg), and megakaryocyte (Meg) colonies wereenumerated.

FIGS. 15A-B. Human donor RBC survival in immunodeficient mice with andwithout Clodronate pre-infusion. A. Representative FACS analysis ofperipheral blood of untreated vs Clodronated treated mouse afterindicated time from RBC transfusion. B. Average % circulating human RBCspost-transfusion mouse peripheral blood. N=3 for each group.

FIGS. 16A-B. Dox-inducible GFP expression in human ESCs. FIG. 16A. Twocomponents of Tet-on vector system (CAG-rtTA and TRE-GFP) wereintroduced into separate alleles of the AAVS1 locus in H9 ESCs usingzinc finger-mediated homologous recombination. Gene X is induced by dox.FIG. 16B. Hematopoietic progenitors produced from the modified H9 ESCswere treated with dox for 0, 2 and 4 days, and assayed for GFPexpression.

FIG. 17 . Dox inducible GATA1 shRNAs in K562 cells. Western blot of K562cells stably transfected with 3 GATA1 shRNAs in the lenti-X Tet-onsystem (Clontech)±dox as indicated.

FIGS. 18A-C. Generation of RHD constructs for donor DNA vector. (FIG.18A) Schematic of first two exons of RHCE in a Rh null iPSC and locationof guide RNA in intron 1 designed for CRISPR-mediated gene editing andthe DNA constructed by inventors to insert by homologous recombination.(FIG. 18B) Detailed schematic of insert created with RHD cDNA (exons2-10 of any given variant), arms of homology, PGK promoter, puromycinresistance cassette and LoxP sites. (FIG. 18C) Donor DNA vector with RHDexons 2-10 that allows simple replacement by variant RHD or RHCE cDNAusing restriction enzymes to generate iPSCs with variant RH (at site ofred arrow). gRNA, guide RNA; SA, splice acceptor; Puro, puromycinresistance cassette; Prom, promoter; pA poly A.

FIGS. 19A-B. Generation of Rh null induced pluripotent stem cells(iPSCs). (FIG. 19A) Schematic of first two exons of RHD and RHCE andlocation of guide RNA designed for CRISPR-mediated gene editing todisrupt Rh protein expression. (FIG. 19B) Flow cytometry of cell surfaceRh expression on red blood cells (RBCs) from donor derived RBCs (left),the untargeted Rh+ parent iPSCs (middle), and the targeted Rh null iPSCs(right) showing no Rh expression on the RBCs from targeted Rh nulliPSCs.

FIG. 20 . Culture conditions for iPSC differentiation into hematopoieticprogenitors and subsequent erythroid differentiation. iPSCs aredifferentiated via an embryoid body culture with the cytokines bonemorphogenic protein 4 (BMP4), vascular endothelial growth factor (VEGF),stem cell factor (SCF), thrombopoietin (TPO), Flt3 ligand (FLT3), andfibroblast growth factor (FGF). Hematopoietic progenitors are harvestedand cultured in liquid erythroid cultures supported by erythropoietin(EPO), SCF, and holotransferrin.

FIGS. 21A-B. Antibody screening with induced pluripotent stem cellderived red blood cells (iRBCs). (FIG. 21A) Patient serum containinganti-e was reacted with donor derived panel red cells or iRBCs in thegel card assay and showed agglutination with all e+ cells and noagglutination with all e− cells, consistent with anti-e antibodypresent. (FIG. 21B) Patient serum containing anti-hrS was reacted withdonor derived panel red cells or iRBCs in the gel card assay and showedagglutination with all hrS+ cells and no agglutination with all hrS−cells.

FIG. 22A. Representative schematic of a CRISPR/CAS9 strategy to generateRh-null induced pluripotent stem cells (iPSCs). The left diagram showstwo target guide RNA to RHCE gene, and the right diagram shows arepresentative FACS plot gated for loss of expression of RhCE.

FIG. 22B. Representative image of recombinant reagent cultured red bloodcells (cRBCs)-derived from iPSCs and donor-derived red blood cells(RBCs).

FIG. 22C. Representative images of the morphology of cRBCs.

FIG. 22D. Representative fluorescence-activated cell sorting (FACS)histogram for Rh-associated glycoprotein (RhAG) expression in originaldonor red blood cells (original RBCs) and two iPSC cRBCs cell lines(iPSC cRBCs B1 and B2).

FIG. 23A. Schematic representation of a combination of amplificationmethods showing that yield of cRBCs can be increased by sequentialexpansion of the stem progenitor and erythroid precursor compartment bysequential use of HSC-expansion, HPC-expansion, and erythroblastexpansion media.

FIG. 23B. Representative diagram of the composition of theHSC-expansion, E-expansion, and E-differentiation media used in theOlivier cRBCs production protocol.

FIG. 23C. Representative table illustrating the yield (mean+/−SD) ofcRBCs obtained with (L1) or without (O1) a HSC-expansion step and with a48-hour pulse in E-expansion prior to culture in HSC-expansionconditions (O2).

FIG. 23D. Representative histogram illustrating the large increase inyield observed in condition O2 as compare to conditions O1 and L1.

FIG. 23E. Representative scatterplots illustrating the number of cellswhen hESC-derived, CB or PB CD34⁺ cells are cultured in conditions L1,O1, or O2.

FIG. 23F. Representative histograms illustrating the number of cRBCsobtained after incubation of CB CD34⁺ cells in L1 or 02 conditions for atotal of 21 days or in O2×3 for a total of 35 days. The L1, O2, and O2×3conditions are symbolically represented by pink, green, and red boxes,respectively, in the histogram. The colors of the boxes correspond tothe media composition (FIG. 23B). The size of the boxes is proportionalto the length of incubation.

FIG. 23G. Erythroblast expansion. Representative dotplot illustratingfold-increase in cell number observed when hESC-derived CD34⁺ cells aregrown in SED conditions (left panel). Weekly fold-increases in cellnumber were observed when hESC-derived CD34⁺ cells were grown in SEDconditions, reached a peak at day 23, and stabilized at about 2-fold perweek (right panel). The composition of the erythroblast expansion (SED)medium is shown in the bottom of the panel.

FIG. 24 . Schematic summary of exemplary methods that can be used totype cRBCs and erythroid precursors.

FIG. 25 . Serologic typing of iPSC-derived red blood cells for non-Rhantigens. Using monoclonal typing reagents for K, k, M, N, S, S, Fya,and Fyb, typing was performed with 500,000 iRBCs per gel card assaycolumn. +/− indicates antigen phenotype as predicted by blood groupgenotype. iPSC-derived RBCs agglutinated and remained at the top of thegel matrix when the antigen was predicted to be expressed (+) and didnot agglutinate and pelleted to bottom of column when antigen waspredicted to be absent (−).

DETAILED DESCRIPTION

As discuss above, transfusion therapies are commonly used to treat awide variety of diseases. Some of these, like Sickle Cell Disease,require repeated transfusions over the lifetime of the patient. As such,the introduction of heterologous blood antigens can result in thedevelopment of immune responses to red blood cells in the transfusedblood. This is particularly problematic when considering the presence ofrare blood antigens that are more likely to be viewed as “foreign” byany given transfusion recipient. The ability to assess the presence ofantibodies to blood antigens, including rare blood antigens, in chronictransfusion recipients, is therefore critical to long-term managed care.

Incompatible transfusions can occur when antibody specificities are notaccurately identified. It is therefore imperative that all antibodiespresent in a patient's serum are identified prior to each transfusion.Due to genetic differences, patients with sickle cell disease (SCD)often experience Rh incompatibilities that do not cause problems in thegeneral population. These cases often require sample referral tospecialized reference laboratories, which significantly delays patientcare and increases costs. Testing at reference laboratories consumesresources, both in reagents and highly skilled labor, and often times,the fine specificity and clinical significance of antibodies cannot beidentified. Without wishing to be bound by theory, the present inventorsdiscovered “universal reagent red blood cells” that enable rapid,cost-saving, and reliable antibody identification in the hospitallaboratory to guide transfusion therapy for highly allo-immunizedpatient populations including those needing chronic transfusion,patients with sickle cell disease, who are mainly of African ancestry,patients with thalassemia who are primarily of Asian ancestry, andpatients receiving cancer immunotherapy drugs which interfere withtesting for blood transfusion.

In particular, the availability of red blood cells (RBCs) lacking highprevalence antigens such as Rh null lines, or expressing Rh variants notfound in natural combinations are particularly relevant because currentdonor-derived RBC reagents cannot resolve these complex antibodyspecificities. These reagent red blood cells described herein enable therapid identification of compatible blood donors, and also providestarting material for large-scale reagent cultured rare red blood cellsfor transfusion. These studies are innovative in that they provideclinical grade reagents needed to test for transfusion compatibilitythat currently do not exist, and they provide an opportunity to producereagent cultured red blood cells for transfusion. Using a number ofdifferent genome-editing techniques in induced pluripotent stem cells(iPSCs) to inactivate blood group genes that encode high prevalence RBCantigens, or to introduce constructs to express variant or altered Rhantigens, will eliminate the need for complex work-ups and have thepotential to streamline and standardize antibody identification testingparticularly for, but not limited to, the ˜100,000 patients with SCD inthe US. Importantly, several of the customized iPSC lines would belife-saving “universal” donors for patients sensitized to numerous bloodgroup antigens who are not able to be transfused with standard bloodtransfusion, and serve a currently unmet clinical need.

More than 15 million RBC transfusions are performed each year in theU.S. making RBC transfusions one of the most important medical therapiesavailable. SCD is the most prevalent monogenic disorder in the world andaffects over 100,000 people in the United States. SCD affects 1/625minorities and 8% carry the Hb S mutation termed sickle-cell trait.

RBC transfusion is a primary treatment for patients with SCD andthalassemia major but development of antibodies directed against foreigndonor RBCs (allo-immunization) is a significant complication thataffects 35% and, in some studies, as high as 65% of chronicallytransfused patients, compared to 2-3% in the general population (Rosseet al., Blood 76: 1431-1437, 1990; and Thompson et al., Br. J. Haematol.153: 121-128, 2011). RBC antigen differences between African Americanpatients and blood donors, who are primarily European American,contribute to the incidence of allo-immunization, as does the largenumber of transfusions. A further complication is that patients whodevelop one antibody are at significantly increased risk for multipleantibodies because of as-yet unknown factors and stimulation of theimmune system. Approximately 50% of patients who make antibodies toforeign RBCs also have auto-antibodies (cross-reactive with their ownRBCs) in the serum (data not shown) (Shirey et al., Transfusion42:1435-1441, 2002).

Distinguishing allo- from auto-antibodies is clinically important fortransfusion but is difficult for routine blood bank laboratories andoften requires referral to a high complexity reference laboratory.Complex multiple adsorptions of the serum with known RBCs, followed byretesting of the serum and elution of the antibodies is often required.These procedures consume vast resources and are laborious, both inreagents and highly skilled technical labor. Allo-immunization oftencauses delays in treatment. In some cases, patients with complex andmultiple antibody specificities are taken off life-benefitingtransfusion protocols due to difficulty in finding compatible RBCsnegative for multiple antigens, or who require matching for highprevalence antigens and rare blood types.

Proper matching of patients and blood products is essential becausetransfusion of incompatible blood or blood products can lead to animmune reaction with hemolysis of the transfused RBCs, and, depending onthe severely of the reaction, lead to pain, fever, anemia, acute kidneyfailure, and/or shock. Delayed transfusion reactions, which occur whensensitized patients produce antibodies following a secondary immunereaction, are associated with RBC destruction, anemia, and jaundice.Some patients experience life-threatening hyper-hemolysis syndrome, apoorly understood phenomena that includes acute lysis of the transfusedcells as well as the patient's own cells termed “bystander” hemolysis(Talano et al., Pediatrics 111: e661-665, 2003).

Without wishing to be bound by theory, the present inventors are able toproduce reagent red blood cells with rare or uncommon blood groupantigen combinations and also cRBCs that lack high prevalence antigens(found on all but rare donor RBCs and determined by blood groupgenotyping). This will address a critical issue in transfusion therapyby providing a standardized and rapid means of distinguishing complexallo-antibody specificities from auto-antibodies in the patient's serum.The production of rare reagent RBCs will provide an important resourcenot currently available to hospital blood banks, and some referenceimmunohematology laboratories, as the lack of reagent RBCs for rapididentification of the antibody specificity leads to significant delaysin determining which donors would be compatible and compromisestransfusion safety.

It is estimated that about 10¹³ cells would be sufficient to coverannual needs for reagent cells lacking high prevalence antigens, andthat reference laboratory testing costs would be decreased by at least70% by replacing the labor-intensive auto- and allo-adsorptions thatoften yield ambiguous results.

There are more than 300 blood group antigens, defined by protein andcarbohydrate polymorphisms on the RBC, reflecting the diversity ofpeople from different ethnic populations. For most transfusiontherapies, matching the patient with the donor for the ABO and RhD bloodgroups is sufficient to avoid eliciting an immune reaction. However,transfusion therapy becomes more complex in chronically transfusedpatients who often produce antibodies against foreign red cell antigens.The causes of the high incidence of allo-immunization in chronicallytransfused patients include the large number of donor RBC exposures,ethnic blood group differences, and possibly chronic inflammation.

Despite attempts to preventively match patients and donors foradditional blood groups (C, E, and K), the incidence ofallo-immunization in chronically transfused patients remains high.Paradoxically, allo-immunized individuals can have RBCs that typepositive for an antigen, but also have the corresponding antibody in theserum. This defies a principal dogma in transfusion medicine, which isthat a patient whose RBCs type positive for an antigen is not at risk ofproducing an antibody to that antigen. This most often occurs in the Rhsystem with D, C, and e antigens. A major cause of this paradox is thatmany patients with SCD have inherited altered RH alleles, which leads tothe recognition of conventional Rh proteins as foreign. In approximately⅓ of these cases, the presence of these antibodies was associated withsignificant destruction of the transfused RBCs (Chou et al., Blood 122:1062-1071, 2013). Other antibodies that are often found in the serum ofpatients with SCD include anti-U, anti-hrS, and anti-hrB, but these areheterogeneous specificities and often are not compatible within eachgroup. RH genotyping can accurately classify the fine specificity and iskey to finding compatible donors. Approximately 2% (U⁻) to 4% (hrS⁻ andhrB⁻) of African-Americans lack these antigens on their RBCs, and aretherefore at risk of making allo-antibodies. These allo-antibodies,which are clinically significant, cannot be distinguished fromauto-antibodies in traditional testing, as they react with all red cellsprovided with current commercial panels.

The present disclosure reports on the development of a panel of modifiedred blood cells that engineered for use in testing the blood oftransfusion patients for antibodies against rare blood antigens. Theseand other aspects of the disclosure are described in detail below.

I. BLOOD GROUPS AND ENGINEERED BLOOD CELLS

A. Blood Groups

The term human blood group systems is defined by International Societyof Blood Transfusion as systems in the human species where cell-surfaceantigens—in particular, those on blood cells—are “controlled at a singlegene locus or by two or more very closely linked homologous genes withlittle or no observable recombination between them,” and include thecommon ABO and Rh-(Rhesus) antigen systems, as well as many others;thirty-six major human systems are identified as of February 2018. Inaddition to the ABO and Rh systems, the antigens expressed on blood cellmembrane surfaces include 346 red blood cell antigens and 33 plateletantigens, as defined serologically. The genetic basis for most of theseantigens lie in 45 red blood cell and 6 platelet genes. An individual,for example, can be AB RhD positive, and at the same time M and Npositive in the MNS system, K positive in the Kell system, and Le^(a) orLe^(b) positive in the Lewis system, where these and many of the systemsare named for patients in whom the corresponding antibodies were firstdetected.

Blood is composed of cells suspended in a liquid called plasma.Suspended in the plasma are three types of cells: Red blood cells thatcarry oxygen; White blood cells that fight infection; and Platelets thatstop bleeding in injuries. The most common type of grouping is the ABO(either uppercase or lowercase) grouping. The varieties of glycoproteincoating on red blood cells divides blood into four groups: A (Aoligosaccharide is present); B (B oligosaccharide is present); AB (A andB oligosaccharides are present); and O (neither A nor B, only theirprecursor H oligosaccharide present). There are subtypes under thisgrouping (listed as A1, A2, A1B or A2B . . . ) some of which are quiterare. Apart from this there is a protein which plays an important partin the grouping of blood. This is called the Rh factor. If this ispresent, the particular blood type is called positive. If it is absent,it is called negative. Thus, there are the following broad categories:

-   -   A1 Negative (A1 moni −ve)    -   A1 Positive (A1 +ve)    -   A1B Negative (A1B −ve)    -   A1B Positive (A1B +ve)    -   A2 Negative (A2 −ve)    -   A2 Positive (A2 +ve)    -   A2B Negative (A2B −ve)    -   A2B Positive (A2B +ve)    -   B Negative (B −ve)    -   B Positive (B +ve)    -   B1 Positive (B1 +ve)    -   O Negative (O −ve)    -   O Positive (O +ve)

In the “ABO” system, (and Rhesus D system) all blood belongs to one offour major groups: A±, B±, AB±, or O±. The presence (+) or absence (−)of the RhD (Rhesus D) antigen is indicated by the plus or minusfollowing the ABO type. But there are more than two hundred minor bloodgroups that can complicate blood transfusions. Many of these are knownas rare blood types. Whereas common blood types are expressed in aletter or two, which may be a plus or a minus, a smaller number ofpeople express their blood type in an extensive series of letters inaddition to their “AB−” type designation. For example, the h/h bloodgroup, also known as Oh or the Bombay blood group, is a rare blood type.

B. Engineered Blood Cells

Provided herein are methods of making reagent or recombinant red bloodcell (e.g., a first reagent red blood cell, a second reagent red bloodcell, or any of the other reagent red blood cells described herein) thatis characterized by the presence of one or more cell surface antigens(e.g., one or more blood group antigens (e.g., any of the blood groupantigens described herein or known in the art)) on its surface. In someembodiments, a reagent red blood cell (e.g., a second reagent red bloodcell) or a recombinant red blood cell can lack at least one cell surfaceantigen (e.g., one or more blood group antigens) that is otherwisepresent on a naturally-occurring red blood cell or another reagent redblood cell (e.g., a first reagent red blood cell).

Induced pluripotent stem cells (iPSCs) are a permanent source of cellsthat can theoretically be used to produce an unlimited number of reagentor recombinant RBCs. Methods of generating a RBC from an iPSC cell areknown in the art, and can be used herein (see, e.g., Example 2). Forexample, a genetic modification (e.g., gene editing to remove or mutatea gene that encodes an antigen) can be performed on a precursor cell(e.g., iPSCs) and the iPSCs differentiated or cultured to yield reagentred blood cells (e.g., any of the reagent red blood cells describedherein). Non-limiting methods for performing gene editing to remove ormutate a gene that encodes an antigen are described herein. Additionalmethods for performing gene editing to remove or mutate a gene thatencodes an antigen are known in the art (e.g., site-specificrecombination).

Some embodiments of these methods can include introducing a nucleic acidthat encodes an antigen (e.g., a blood group antigen) into a red bloodcell (e.g., a reagent red blood cell) or into a precursor cell (e.g.,iPSCs) that is cultured or differentiated into a reagent red blood cell.Exemplary methods for introducing a nucleic acid that encodes an antigeninto a cell are described herein. Additional methods for introducing anucleic acid that encodes an antigen into a cell are known in the art.

Methods of culturing cells are well known in the art. Cells can bemaintained in vitro under conditions that favor proliferation,differentiation, and growth. Briefly, cells can be cultured bycontacting a cell (e.g., any cell) with a cell culture medium thatincludes the necessary growth factors and supplements to support cellviability and growth. Additional exemplary methods for generating areagent red blood cell are described below.

Non-limiting examples of reagent red blood cells that can be produced bythe methods described herein are listed in Table 1.4 below.

TABLE 1.4 Exemplary Panel of Reagent Cultured Red Blood Cells RelevantGenotype RBC phenotype Antibody detection Rh null No RHD, inactive D−,C−, E−, c−, e− Identify antibodies against any high RHCE (no Rhantigens) prevalence antigens in Rh system D−− Inactive RHCE D+, C−, E−,c−, e− Identify antibodies to RHCE (no RhCE antigens) U−S−s− InactiveGYB D−, U−, S−, s− Identify antibodies against high prevalence Uantigen, and against S/s antigens hrB−, RHCE*ce(733G) D−, hrB−, VS+Identify antibodies against high VS+ prevalence hrB antigen (−)reaction), or to low prevalence VS antigen ((+) reaction) hrB−,RHCE*ce(48C, D−, hrB−, hrS− Identify specificity antibodies against hrS−667T) high prevalence RHCE (hrB vs hrS) antigens ((−) reaction) Rh nullRHD*DIVa on Rh− D+, C−, E−, c−, e− Identify antibodies to this antigenGo(a)+ nullbackground Go(a)+ which is unique to African AmericansRh−null RHD*DIIIa on Rh− D+, C−, E−, c−, e− Identify antibodies to thisantigen DAK+ null background DAK+ which is unique to African AmericansDo null Inactive ART D−, Doa−, Dob− Identify antibodies against highprevalence Do and HY antigens. Most useful as a future transfusionproduct. Lua−b− EKLF or LU inactive For testing patients on anti-CD38therapy CD47 CD47 null For testing patients on anti-CD47 null therapy

In some examples, the reagent red blood cells provided herein have asurface phenotype that does not naturally occur in a human or othermammal. In some embodiments, reagent red blood cells that arecharacterized, at least in part, by a D⁻, C⁻, E⁻, c⁻, e⁻ surfacephenotype are not naturally-occurring in a human or other mammal. Insome embodiments, reagent red blood cells that are characterized, atleast in part, by a D⁺, C⁻, E⁻, c⁻, e⁻ surface phenotype are notnaturally-occurring in a human or other mammal. In some embodiments,reagent red blood cells that are characterized, at least in part, by aD⁺, C⁻, E⁻, c⁻, e⁻, Go(a)⁺ surface phenotype are not naturally-occurringin a human or other mammal. In some embodiments, reagent red blood cellsthat are characterized, at least in part, by a D⁺, C⁻, E⁻, c⁻, e⁻, DAK⁺surface phenotype are not naturally-occurring in a human or othermammal. In some embodiments, reagent red blood cells that arecharacterized, at least in part, by a D⁻, Doa⁻, Dob⁻ surface phenotypeare not naturally-occurring in a human or other mammal. In someembodiments, reagent red blood cells that are characterized, at least inpart, by a CD47⁻ surface phenotype are not naturally-occurring in ahuman or other mammal.

In some embodiments, a reagent red blood cell that is characterized, atleast in part, by the absence of a C antigen, an E antigen, a c antigen,an e antigen, a U antigen, a S antigen, a s antigen, a hrB antigen, aLua antigen, a Lub antigen, and a CD47 antigen on its surface are notnaturally-occurring in a human or other mammal.

In some embodiments, reagent red blood cells that are characterized, atleast in part, by a D⁻, U⁻, S⁻, s⁻ surface phenotype, which is presentin less than 1% (e.g., less than 0.5%, less than 0.2%, less than 0.1%,or less than 0.05%) of patients. In some embodiments, reagent red bloodcells that are characterized, at least in part, by a D⁻, hrB⁻, VS⁺phenotype, which is present in less than 1% (e.g., less than 0.5%, lessthan 0.2%, less than 0.1%, or less than 0.05%) of patients. In someembodiments, reagent red blood cells that are characterized, at least inpart, by a Lua⁻, b⁻ phenotype, which is present in less than 1% (e.g.,less than 0.5%, less than 0.2%, less than 0.1%, or less than 0.05%) ofpatients.

Additional embodiments of exemplary reagent red blood cells providedherein can have a surface phenotype characterized, at least in part, byone of the following, or a combination of two or more of any of thefollowing:

(i) D−, C−, E−, c−, e−;

(ii) D+, C−, E−, c−, e−;

(iii) D−, U−, S−, s−;

(iv) D−, hrB−, VS+;

(v) D−, hrB−, hrS−;

(vi) C−, E−, c−, e−;

(vii) D+, C−, E−, c−, e−, Go(a)+;

(viii) D+, C−, E−, c−, e−, DAK+;

(ix) D−, Doa−, Dob−;

(x) Lua− b−; and

(xi) CD47−.

For example, a reagent red blood can have a surface phenotypecharacterized by D−, C−, E−, c−, e−, Doa− and Dob−. In some instances, areagent red blood can have a surface phenotype characterized by D−, C−,E−, c−, e−, Lua− and b−. The specific examples of reagent red bloodcells described herein are exemplary. As can be appreciated, manyadditional reagent red blood cells having different surface phenotypescan be generated using the methods described herein. For example, any ofthe compositions, kits, and methods described herein can include orinclude the use of at least 2 (e.g., at least 3, at least 4, at least 5,at least 6, at least 7, at least 8, at least 9, at least 10, at least11, at least 12, at least 13, at least 14, at least 15, at least 16, atleast 17, at least 18, at least 19, at least 20, at least 21, at least22, at least 23, at least 24, at least 25, at least 26, at least 27, atleast 28, at least 29, at least 30, at least 31, at least 32, at least33, at least 34, at least 35, at least 36, at least 37, at least 38, atleast 39, at least 40, at least 41, at least 42, at least 43, at least44, at least 45, at least 46, at least 47, at least 48, at least 49, atleast 50, at least 51, at least 52, at least 53, at least 54, at least55, at least 56, at least 57, at least 58, at least 59, at least 60, atleast 61, at least 62, at least 63, at least 64, at least 65, at least66, at least 67, at least 68, at least 69, at least 70, at least 71, atleast 72, at least 73, at least 74, at least 75, at least 76, at least77, at least 78, at least 79, at least 80, at least 81, at least 82, atleast 83, at least 84, at least 85, at least 86, at least 87, at least88, at least 89, at least 90, at least 91, at least 92, at least 93, atleast 94, at least 95, at least 96, at least 97, at least 98, at least99, at least 100, at least 110, at least 115, at least 120, at least125, at least 130, at least 135, at least 140, at least 145, at least150, at least 155, at least 160, at least 165, at least 170, at least175, at least 180, at least 185, at least 190, at least 195, or at least200) different reagent red blood cells (e.g., any of the exemplaryreagent red blood cells described herein, including, e.g., the firstreagent red blood cell and the second reagent red blood cell), whereineach of the reagent red blood cells had a different surface phenotypefrom any of the other reagent red blood cells. For example, any of thecompositions, kits, and methods described herein can include or includethe use of 2 to about 200 (e.g., 2 to about 195, 2 to about 190, 2 toabout 185, 2 to about 180, 2 to about 175, 2 to about 170, 2 to about165, 2 to about 160, 2 to about 155, 2 to about 150, 2 to about 145, 2to about 140, 2 to about 135, 2 to about 130, 2 to about 125, 2 to about120, 2 to about 115, 2 to about 110, 2 to about 105, 2 to about 100, 2to about 95, 2 to about 90, 2 to about 85, 2 to about 80, 2 to about 75,2 to about 70, 2 to about 65, 2 to about 60, 2 to about 55, 2 to about50, 2 to about 45, 2 to about 40, 2 to about 35, 2 to about 30, 2 toabout 25, 2 to about 20, 2 to about 18, 2 to about 16, 2 to about 14, 2to about 12, 2 to about 10, 2 to about 8, 2 to about 6, 2 to about 5,about 3 to about 200, about 3 to about 195, about 3 to about 190, about3 to about 185, about 3 to about 180, about 3 to about 175, about 3 toabout 170, about 3 to about 165, about 3 to about 160, about 3 to about155, about 3 to about 150, about 3 to about 145, about 3 to about 140,about 3 to about 135, about 3 to about 130, about 3 to about 125, about3 to about 120, about 3 to about 115, about 3 to about 110, about 3 toabout 105, about 3 to about 100, about 3 to about 95, about 3 to about90, about 3 to about 85, about 3 to about 80, about 3 to about 75, about3 to about 70, about 3 to about 65, about 3 to about 60, about 3 toabout 55, about 3 to about 50, about 3 to about 45, about 3 to about 40,about 3 to about 35, about 3 to about 30, about 3 to about 25, about 3to about 20, about 3 to about 18, about 3 to about 16, about 3 to about14, about 3 to about 12, about 3 to about 10, about 3 to about 8, about3 to about 6, about 3 to about 5, about 4 to about 200, about 4 to about195, about 4 to about 190, about 4 to about 185, about 4 to about 180,about 4 to about 175, about 4 to about 170, about 4 to about 165, about4 to about 160, about 4 to about 155, about 4 to about 150, about 4 toabout 145, about 4 to about 140, about 4 to about 135, about 4 to about130, about 4 to about 125, about 4 to about 120, about 4 to about 115,about 4 to about 110, about 4 to about 105, about 4 to about 100, about4 to about 95, about 4 to about 90, about 4 to about 85, about 4 toabout 80, about 4 to about 75, about 4 to about 70, about 4 to about 65,about 4 to about 60, about 4 to about 55, about 4 to about 50, about 4to about 45, about 4 to about 40, about 4 to about 35, about 4 to about30, about 4 to about 25, about 4 to about 20, about 4 to about 18, about4 to about 16, about 4 to about 14, about 4 to about 12, about 4 toabout 10, about 4 to about 8, about 4 to about 6, about 5 to about 200,about 5 to about 195, about 5 to about 190, about 5 to about 185, about5 to about 180, about 5 to about 175, about 5 to about 170, about 5 toabout 165, about 5 to about 160, about 5 to about 155, about 5 to about150, about 5 to about 145, about 5 to about 140, about 5 to about 135,about 5 to about 130, about 5 to about 125, about 5 to about 120, about5 to about 115, about 5 to about 110, about 5 to about 105, about 5 toabout 100, about 5 to about 95, about 5 to about 90, about 5 to about85, about 5 to about 80, about 5 to about 75, about 5 to about 70, about5 to about 65, about 5 to about 60, about 5 to about 55, about 5 toabout 50, about 5 to about 45, about 5 to about 40, about 5 to about 35,about 5 to about 30, about 5 to about 25, about 5 to about 20, about 5to about 18, about 5 to about 16, about 5 to about 14, about 5 to about12, about 5 to about 10, about 5 to about 8, about 6 to about 200, about6 to about 195, about 6 to about 190, about 6 to about 185, about 6 toabout 180, about 6 to about 175, about 6 to about 170, about 6 to about165, about 6 to about 160, about 6 to about 155, about 6 to about 150,about 6 to about 145, about 6 to about 140, about 6 to about 135, about6 to about 130, about 6 to about 125, about 6 to about 120, about 6 toabout 115, about 6 to about 110, about 6 to about 105, about 6 to about100, about 6 to about 95, about 6 to about 90, about 6 to about 85,about 6 to about 80, about 6 to about 75, about 6 to about 70, about 6to about 65, about 6 to about 60, about 6 to about 55, about 6 to about50, about 6 to about 45, about 6 to about 40, about 6 to about 35, about6 to about 30, about 6 to about 25, about 6 to about 20, about 6 toabout 18, about 6 to about 16, about 6 to about 14, about 6 to about 12,about 6 to about 10, about 6 to about 8, about 8 to about 200, about 8to about 195, about 8 to about 190, about 8 to about 185, about 8 toabout 180, about 8 to about 175, about 8 to about 170, about 8 to about165, about 8 to about 160, about 8 to about 155, about 8 to about 150,about 8 to about 145, about 8 to about 140, about 8 to about 135, about8 to about 130, about 8 to about 125, about 8 to about 120, about 8 toabout 115, about 8 to about 110, about 8 to about 105, about 8 to about100, about 8 to about 95, about 8 to about 90, about 8 to about 85,about 8 to about 80, about 8 to about 75, about 8 to about 70, about 8to about 65, about 8 to about 60, about 8 to about 55, about 8 to about50, about 8 to about 45, about 8 to about 40, about 8 to about 35, about8 to about 30, about 8 to about 25, about 8 to about 20, about 8 toabout 18, about 8 to about 16, about 8 to about 14, about 8 to about 12,about 8 to about 10, about 10 to about 200, about 10 to about 195, about10 to about 190, about 10 to about 185, about 10 to about 180, about 10to about 175, about 10 to about 170, about 10 to about 165, about 10 toabout 160, about 10 to about 155, about 10 to about 150, about 10 toabout 145, about 10 to about 140, about 10 to about 135, about 10 toabout 130, about 10 to about 125, about 10 to about 120, about 10 toabout 115, about 10 to about 110, about 10 to about 105, about 10 toabout 100, about 10 to about 95, about 10 to about 90, about 10 to about85, about 10 to about 80, about 10 to about 75, about 10 to about 70,about 10 to about 65, about 10 to about 60, about 10 to about 55, about10 to about 50, about 10 to about 45, about 10 to about 40, about 10 toabout 35, about 10 to about 30, about 10 to about 25, about 10 to about20, about 10 to about 18, about 10 to about 16, about 10 to about 14,about 10 to about 12, about 12 to about 200, about 12 to about 195,about 12 to about 190, about 12 to about 185, about 12 to about 180,about 12 to about 175, about 12 to about 170, about 12 to about 165,about 12 to about 160, about 12 to about 155, about 12 to about 150,about 12 to about 145, about 12 to about 140, about 12 to about 135,about 12 to about 130, about 12 to about 125, about 12 to about 120,about 12 to about 115, about 12 to about 110, about 12 to about 105,about 12 to about 100, about 12 to about 95, about 12 to about 90, about12 to about 85, about 12 to about 80, about 12 to about 75, about 12 toabout 70, about 12 to about 65, about 12 to about 60, about 12 to about55, about 12 to about 50, about 12 to about 45, about 12 to about 40,about 12 to about 35, about 12 to about 30, about 12 to about 25, about12 to about 20, about 12 to about 18, about 12 to about 16, about 12 toabout 14, about 14 to about 200, about 14 to about 195, about 14 toabout 190, about 14 to about 185, about 14 to about 180, about 14 toabout 175, about 14 to about 170, about 14 to about 165, about 14 toabout 160, about 14 to about 155, about 14 to about 150, about 14 toabout 145, about 14 to about 140, about 14 to about 135, about 14 toabout 130, about 14 to about 125, about 14 to about 120, about 14 toabout 115, about 14 to about 110, about 14 to about 105, about 14 toabout 100, about 14 to about 95, about 14 to about 90, about 14 to about85, about 14 to about 80, about 14 to about 75, about 14 to about 70,about 14 to about 65, about 14 to about 60, about 14 to about 55, about14 to about 50, about 14 to about 45, about 14 to about 40, about 14 toabout 35, about 14 to about 30, about 14 to about 25, about 14 to about20, about 14 to about 18, about 14 to about 16, about 16 to about 200,about 16 to about 195, about 16 to about 190, about 16 to about 185,about 16 to about 180, about 16 to about 175, about 16 to about 170,about 16 to about 165, about 16 to about 160, about 16 to about 155,about 16 to about 150, about 16 to about 145, about 16 to about 140,about 16 to about 135, about 16 to about 130, about 16 to about 125,about 16 to about 120, about 16 to about 115, about 16 to about 110,about 16 to about 105, about 16 to about 100, about 16 to about 95,about 16 to about 90, about 16 to about 85, about 16 to about 80, about16 to about 75, about 16 to about 70, about 16 to about 65, about 16 toabout 60, about 16 to about 55, about 16 to about 50, about 16 to about45, about 16 to about 40, about 16 to about 35, about 16 to about 30,about 16 to about 25, about 16 to about 20, about 16 to about 18, about18 to about 200, about 18 to about 195, about 18 to about 190, about 18to about 185, about 18 to about 180, about 18 to about 175, about 18 toabout 170, about 18 to about 165, about 18 to about 160, about 18 toabout 155, about 18 to about 150, about 18 to about 145, about 18 toabout 140, about 18 to about 135, about 18 to about 130, about 18 toabout 125, about 18 to about 120, about 18 to about 115, about 18 toabout 110, about 18 to about 105, about 18 to about 100, about 18 toabout 95, about 18 to about 90, about 18 to about 85, about 18 to about80, about 18 to about 75, about 18 to about 70, about 18 to about 65,about 18 to about 60, about 18 to about 55, about 18 to about 50, about18 to about 45, about 18 to about 40, about 18 to about 35, about 18 toabout 30, about 18 to about 25, about 18 to about 20, about 20 to about200, about 20 to about 195, about 20 to about 190, about 20 to about185, about 20 to about 180, about 20 to about 175, about 20 to about170, about 20 to about 165, about 20 to about 160, about 20 to about155, about 20 to about 150, about 20 to about 145, about 20 to about140, about 20 to about 135, about 20 to about 130, about 20 to about125, about 20 to about 120, about 20 to about 115, about 20 to about110, about 20 to about 105, about 20 to about 100, about 20 to about 95,about 20 to about 90, about 20 to about 85, about 20 to about 80, about20 to about 75, about 20 to about 70, about 20 to about 65, about 20 toabout 60, about 20 to about 55, about 20 to about 50, about 20 to about45, about 20 to about 40, about 20 to about 35, about 20 to about 30,about 20 to about 25, about 25 to about 200, about 25 to about 195,about 25 to about 190, about 25 to about 185, about 25 to about 180,about 25 to about 175, about 25 to about 170, about 25 to about 165,about 25 to about 160, about 25 to about 155, about 25 to about 150,about 25 to about 145, about 25 to about 140, about 25 to about 135,about 25 to about 130, about 25 to about 125, about 25 to about 120,about 25 to about 115, about 25 to about 110, about 25 to about 105,about 25 to about 100, about 25 to about 95, about 25 to about 90, about25 to about 85, about 25 to about 80, about 25 to about 75, about 25 toabout 70, about 25 to about 65, about 25 to about 60, about 25 to about55, about 25 to about 50, about 25 to about 45, about 25 to about 40,about 25 to about 35, about 25 to about 30, about 30 to about 200, about30 to about 195, about 30 to about 190, about 30 to about 185, about 30to about 180, about 30 to about 175, about 30 to about 170, about 30 toabout 165, about 30 to about 160, about 30 to about 155, about 30 toabout 150, about 30 to about 145, about 30 to about 140, about 30 toabout 135, about 30 to about 130, about 30 to about 125, about 30 toabout 120, about 30 to about 115, about 30 to about 110, about 30 toabout 105, about 30 to about 100, about 30 to about 95, about 30 toabout 90, about 30 to about 85, about 30 to about 80, about 30 to about75, about 30 to about 70, about 30 to about 65, about 30 to about 60,about 30 to about 55, about 30 to about 50, about 30 to about 45, about30 to about 40, about 30 to about 35, about 35 to about 200, about 35 toabout 195, about 35 to about 190, about 35 to about 185, about 35 toabout 180, about 35 to about 175, about 35 to about 170, about 35 toabout 165, about 35 to about 160, about 35 to about 155, about 35 toabout 150, about 35 to about 145, about 35 to about 140, about 35 toabout 135, about 35 to about 130, about 35 to about 125, about 35 toabout 120, about 35 to about 115, about 35 to about 110, about 35 toabout 105, about 35 to about 100, about 35 to about 95, about 35 toabout 90, about 35 to about 85, about 35 to about 80, about 35 to about75, about 35 to about 70, about 35 to about 65, about 35 to about 60,about 35 to about 55, about 35 to about 50, about 35 to about 45, about35 to about 40, about 40 to about 200, about 40 to about 195, about 40to about 190, about 40 to about 185, about 40 to about 180, about 40 toabout 175, about 40 to about 170, about 40 to about 165, about 40 toabout 160, about 40 to about 155, about 40 to about 150, about 40 toabout 145, about 40 to about 140, about 40 to about 135, about 40 toabout 130, about 40 to about 125, about 40 to about 120, about 40 toabout 115, about 40 to about 110, about 40 to about 105, about 40 toabout 100, about 40 to about 95, about 40 to about 90, about 40 to about85, about 40 to about 80, about 40 to about 75, about 40 to about 70,about 40 to about 65, about 40 to about 60, about 40 to about 55, about40 to about 50, about 40 to about 45, about 45 to about 200, about 45 toabout 195, about 45 to about 190, about 45 to about 185, about 45 toabout 180, about 45 to about 175, about 45 to about 170, about 45 toabout 165, about 45 to about 160, about 45 to about 155, about 45 toabout 150, about 45 to about 145, about 45 to about 140, about 45 toabout 135, about 45 to about 130, about 45 to about 125, about 45 toabout 120, about 45 to about 115, about 45 to about 110, about 45 toabout 105, about 45 to about 100, about 45 to about 95, about 45 toabout 90, about 45 to about 85, about 45 to about 80, about 45 to about75, about 45 to about 70, about 45 to about 65, about 45 to about 60,about 45 to about 55, about 45 to about 50, about 50 to about 200, about50 to about 195, about 50 to about 190, about 50 to about 185, about 50to about 180, about 50 to about 175, about 50 to about 170, about 50 toabout 165, about 50 to about 160, about 50 to about 155, about 50 toabout 150, about 50 to about 145, about 50 to about 140, about 50 toabout 135, about 50 to about 130, about 50 to about 125, about 50 toabout 120, about 50 to about 115, about 50 to about 110, about 50 toabout 105, about 50 to about 100, about 50 to about 95, about 50 toabout 90, about 50 to about 85, about 50 to about 80, about 50 to about75, about 50 to about 70, about 50 to about 65, about 50 to about 60,about 50 to about 55, about 55 to about 200, about 55 to about 195,about 55 to about 190, about 55 to about 185, about 55 to about 180,about 55 to about 175, about 55 to about 170, about 55 to about 165,about 55 to about 160, about 55 to about 155, about 55 to about 150,about 55 to about 145, about 55 to about 140, about 55 to about 135,about 55 to about 130, about 55 to about 125, about 55 to about 120,about 55 to about 115, about 55 to about 110, about 55 to about 105,about 55 to about 100, about 55 to about 95, about 55 to about 90, about55 to about 85, about 55 to about 80, about 55 to about 75, about 55 toabout 70, about 55 to about 65, about 55 to about 60, about 60 to about200, about 60 to about 195, about 60 to about 190, about 60 to about185, about 60 to about 180, about 60 to about 175, about 60 to about170, about 60 to about 165, about 60 to about 160, about 60 to about155, about 60 to about 150, about 60 to about 145, about 60 to about140, about 60 to about 135, about 60 to about 130, about 60 to about125, about 60 to about 120, about 60 to about 115, about 60 to about110, about 60 to about 105, about 60 to about 100, about 60 to about 95,about 60 to about 90, about 60 to about 85, about 60 to about 80, about60 to about 75, about 60 to about 70, about 60 to about 65, about 65 toabout 200, about 65 to about 195, about 65 to about 190, about 65 toabout 185, about 65 to about 180, about 65 to about 175, about 65 toabout 170, about 65 to about 165, about 65 to about 160, about 65 toabout 155, about 65 to about 150, about 65 to about 145, about 65 toabout 140, about 65 to about 135, about 65 to about 130, about 65 toabout 125, about 65 to about 120, about 65 to about 115, about 65 toabout 110, about 65 to about 105, about 65 to about 100, about 65 toabout 95, about 65 to about 90, about 65 to about 85, about 65 to about80, about 65 to about 75, about 65 to about 70, about 70 to about 200,about 70 to about 195, about 70 to about 190, about 70 to about 185,about 70 to about 180, about 70 to about 175, about 70 to about 170,about 70 to about 165, about 70 to about 160, about 70 to about 155,about 70 to about 150, about 70 to about 145, about 70 to about 140,about 70 to about 135, about 70 to about 130, about 70 to about 125,about 70 to about 120, about 70 to about 115, about 70 to about 110,about 70 to about 105, about 70 to about 100, about 70 to about 95,about 70 to about 90, about 70 to about 85, about 70 to about 80, about70 to about 75, about 75 to about 200, about 75 to about 195, about 75to about 190, about 75 to about 185, about 75 to about 180, about 75 toabout 175, about 75 to about 170, about 75 to about 165, about 75 toabout 160, about 75 to about 155, about 75 to about 150, about 75 toabout 145, about 75 to about 140, about 75 to about 135, about 75 toabout 130, about 75 to about 125, about 75 to about 120, about 75 toabout 115, about 75 to about 110, about 75 to about 105, about 75 toabout 100, about 75 to about 95, about 75 to about 90, about 75 to about85, about 75 to about 80, about 80 to about 200, about 80 to about 195,about 80 to about 190, about 80 to about 185, about 80 to about 180,about 80 to about 175, about 80 to about 170, about 80 to about 165,about 80 to about 160, about 80 to about 155, about 80 to about 150,about 80 to about 145, about 80 to about 140, about 80 to about 135,about 80 to about 130, about 80 to about 125, about 80 to about 120,about 80 to about 115, about 80 to about 110, about 80 to about 105,about 80 to about 100, about 80 to about 95, about 80 to about 90, about80 to about 85, about 85 to about 200, about 85 to about 195, about 85to about 190, about 85 to about 185, about 85 to about 180, about 85 toabout 175, about 85 to about 170, about 85 to about 165, about 85 toabout 160, about 85 to about 155, about 85 to about 150, about 85 toabout 145, about 85 to about 140, about 85 to about 135, about 85 toabout 130, about 85 to about 125, about 85 to about 120, about 85 toabout 115, about 85 to about 110, about 85 to about 105, about 85 toabout 100, about 85 to about 95, about 85 to about 90, about 90 to about200, about 90 to about 195, about 90 to about 190, about 90 to about185, about 90 to about 180, about 90 to about 175, about 90 to about170, about 90 to about 165, about 90 to about 160, about 90 to about155, about 90 to about 150, about 90 to about 145, about 90 to about140, about 90 to about 135, about 90 to about 130, about 90 to about125, about 90 to about 120, about 90 to about 115, about 90 to about110, about 90 to about 105, about 90 to about 100, about 90 to about 95,about 95 to about 200, about 95 to about 195, about 95 to about 190,about 95 to about 185, about 95 to about 180, about 95 to about 175,about 95 to about 170, about 95 to about 165, about 95 to about 160,about 95 to about 155, about 95 to about 150, about 95 to about 145,about 95 to about 140, about 95 to about 135, about 95 to about 130,about 95 to about 125, about 95 to about 120, about 95 to about 115,about 95 to about 110, about 95 to about 105, about 95 to about 100,about 100 to about 200, about 100 to about 195, about 100 to about 190,about 100 to about 185, about 100 to about 180, about 100 to about 175,about 100 to about 170, about 100 to about 165, about 100 to about 160,about 100 to about 155, about 100 to about 150, about 100 to about 145,about 100 to about 140, about 100 to about 135, about 100 to about 130,about 100 to about 125, about 100 to about 120, about 100 to about 115,about 100 to about 110, about 100 to about 105, about 105 to about 200,about 105 to about 195, about 105 to about 190, about 105 to about 185,about 105 to about 180, about 105 to about 175, about 105 to about 170,about 105 to about 165, about 105 to about 160, about 105 to about 155,about 105 to about 150, about 105 to about 145, about 105 to about 140,about 105 to about 135, about 105 to about 130, about 105 to about 125,about 105 to about 120, about 105 to about 115, about 105 to about 110,about 110 to about 200, about 110 to about 195, about 110 to about 190,about 110 to about 185, about 110 to about 180, about 110 to about 175,about 110 to about 170, about 110 to about 165, about 110 to about 160,about 110 to about 155, about 110 to about 150, about 110 to about 145,about 110 to about 140, about 110 to about 135, about 110 to about 130,about 110 to about 125, about 110 to about 120, about 110 to about 115,about 115 to about 200, about 115 to about 195, about 115 to about 190,about 115 to about 185, about 115 to about 180, about 115 to about 175,about 115 to about 170, about 115 to about 165, about 115 to about 160,about 115 to about 155, about 115 to about 150, about 115 to about 145,about 115 to about 140, about 115 to about 135, about 115 to about 130,about 115 to about 125, about 115 to about 120, about 120 to about 200,about 120 to about 195, about 120 to about 190, about 120 to about 185,about 120 to about 180, about 120 to about 175, about 120 to about 170,about 120 to about 165, about 120 to about 160, about 120 to about 155,about 120 to about 150, about 120 to about 145, about 120 to about 140,about 120 to about 135, about 120 to about 130, about 120 to about 125,about 125 to about 200, about 125 to about 195, about 125 to about 190,about 125 to about 185, about 125 to about 180, about 125 to about 175,about 125 to about 170, about 125 to about 165, about 125 to about 160,about 125 to about 155, about 125 to about 150, about 125 to about 145,about 125 to about 140, about 125 to about 135, about 125 to about 130,about 130 to about 200, about 130 to about 195, about 130 to about 190,about 130 to about 185, about 130 to about 180, about 130 to about 175,about 130 to about 170, about 130 to about 165, about 130 to about 160,about 130 to about 155, about 130 to about 150, about 130 to about 145,about 130 to about 140, about 130 to about 135, about 135 to about 200,about 135 to about 195, about 135 to about 190, about 135 to about 185,about 135 to about 180, about 135 to about 175, about 135 to about 170,about 135 to about 165, about 135 to about 160, about 135 to about 155,about 135 to about 150, about 135 to about 145, about 135 to about 140,about 140 to about 200, about 140 to about 195, about 140 to about 190,about 140 to about 185, about 140 to about 180, about 140 to about 175,about 140 to about 170, about 140 to about 165, about 140 to about 160,about 140 to about 155, about 140 to about 150, about 140 to about 145,about 145 to about 200, about 145 to about 195, about 145 to about 190,about 145 to about 185, about 145 to about 180, about 145 to about 175,about 145 to about 170, about 145 to about 165, about 145 to about 160,about 145 to about 155, about 145 to about 150, about 150 to about 200,about 150 to about 195, about 150 to about 190, about 150 to about 185,about 150 to about 180, about 150 to about 175, about 150 to about 170,about 150 to about 165, about 150 to about 160, about 150 to about 155,about 155 to about 200, about 155 to about 195, about 155 to about 190,about 155 to about 185, about 155 to about 180, about 155 to about 175,about 155 to about 170, about 155 to about 165, about 155 to about 160,about 160 to about 200, about 160 to about 195, about 160 to about 190,about 160 to about 185, about 160 to about 180, about 160 to about 175,about 160 to about 170, about 160 to about 165, about 165 to about 200,about 165 to about 195, about 165 to about 190, about 165 to about 185,about 165 to about 180, about 165 to about 175, about 165 to about 170,about 170 to about 200, about 170 to about 195, about 170 to about 190,about 170 to about 185, about 170 to about 180, about 170 to about 175,about 175 to about 200, about 175 to about 195, about 175 to about 190,about 175 to about 185, about 175 to about 180, about 180 to about 200,about 180 to about 195, about 180 to about 190, about 180 to about 185,about 185 to about 200, about 185 to about 195, about 185 to about 190,about 190 to about 200, about 190 to about 195, or about 195 to about200) different reagent red blood cells (e.g., any of the exemplaryreagent red blood cells described herein, including, e.g., the firstreagent red blood cell and the second reagent red blood cell), whereineach of the reagent red blood cells had a different surface phenotypefrom any of the other reagent red blood cells.

Additional reagent red blood cells can be generated using any of themethods described herein, e.g., a recombinant red blood cell having asurface phenotype lacking at least one (e.g., 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) antigen on its surface(e.g., any of the blood group antigens described herein or known in theart in any combination).

In some embodiments of any of the reagent red blood cells, the reagentred blood cell is a recombinant group O cell.

In some embodiments of any of the test kits described herein, the kitincludes at least one reagent red blood cell that has a surfacephenotype that is not naturally-occurring in a human or other mammal.

Also provided are recombinant red blood cells, wherein each recombinantred blood cell is characterized by the absence of at least one or more(e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) cell surface antigens on itssurface selected from the group of: a C antigen, an E antigen, a cantigen, an e antigen, a U antigen, an S antigen, an s antigen, an hrBantigen, a Lua antigen, a Lub antigen and a CD47 antigen. Someembodiments of these recombinant red blood cells are furthercharacterized by the absence of a D antigen on its cell surface. Otherembodiments of these recombinant red blood cells are furthercharacterized by the presence of a D antigen on its cell surface. Someembodiments of these recombinant red blood cells are furthercharacterized by the presence of a Go(a) antigen on its cell surface.Some embodiments of these recombinant red blood cells are furthercharacterized by the presence of a DAK antigen on its cell surface. Someembodiments of these recombinant red blood cells are characterized bythe absence of a Doa antigen and a Dob antigen on its cell surface.

The surface phenotype of a reagent red blood cell described herein canbe determined or confirmed using one or more antibodies thatspecifically recognize an antigen that is presented on the surface ofthe reagent red blood cell (e.g., using fluorescence-assisted cellsorting and immunofluorescence or agglutination).

The following references described the engineering of cells, includingstem cells, to have altered blood group antigens: U.S. Pat. No.5,811,130; WO2016085934; WO2015032340; CA2516123; WO2015118780; U.S.Pat. Nos. 9,200,253; 9,255,248; 9,169,462.

II. SICKLE CELL DISEASE AND TRANSFUSION THERAPY

A. Sickle Cell Disease

Sickle-cell disease (SCD) is a group of blood disorders typicallyinherited from a person's parents. The most common type is known assickle-cell anaemia (SCA). It results in an abnormality in theoxygen-carrying protein hemoglobin (hemoglobin S) found in red bloodcells. This leads to a rigid, sickle-like shape under certaincircumstances. Problems in sickle cell disease typically begin around 5to 6 months of age. A number of health problems may develop, such asattacks of pain (“sickle-cell crisis”), anemia, swelling in the handsand feet, bacterial infections, and stroke. Long term pain may developas people get older. The average life expectancy in the developed worldis 40 to 60 years.

Sickle-cell disease occurs when a person inherits two abnormal copies ofthe hemoglobin gene, one from each parent. This gene occurs inchromosome 11. Several subtypes exist, depending on the exact mutationin each hemoglobin gene. An attack can be set off by temperaturechanges, stress, dehydration, and high altitude. A person with a singleabnormal copy does not usually have symptoms and is said to havesickle-cell trait. Such people are also referred to as carriers.Diagnosis is by a blood test and some countries test all babies at birthfor the disease. Diagnosis is also possible during pregnancy.

The care of people with sickle-cell disease may include infectionprevention with vaccination and antibiotics, high fluid intake, folicacid supplementation, and pain medication. Other measures may includeblood transfusion, and the medication hydroxycarbamide (hydroxyurea). Asmall proportion of people can be cured by a transplant of bone marrowcells. As of 2015 about 4.4 million people have sickle-cell diseasewhile an additional 43 million have sickle-cell trait. About 80% ofsickle-cell disease cases are believed to occur in sub-Saharan Africa.It also occurs relatively frequently in parts of India, the Arabianpeninsula, and among people of African origin living in other parts ofthe world. In 2015, it resulted in about 114,800 deaths.

Signs of sickle cell disease usually begin in early childhood. Theseverity of symptoms can vary from person to person, Sickle-cell diseasemay lead to various acute and chronic complications, several of whichhave a high mortality rate.

The terms “sickle-cell crisis” or “sickling crisis” may be used todescribe several independent acute conditions occurring in patients withSCD. SCD results in anemia and crises that could be of many typesincluding the vaso-occlusive crisis, aplastic crisis, sequestrationcrisis, haemolytic crisis, and others. Most episodes of sickle-cellcrises last between five and seven days. “Although infection,dehydration, and acidosis (all of which favor sickling) can act astriggers, in most instances, no predisposing cause is identified.”

The vaso-occlusive crisis is caused by sickle-shaped red blood cellsthat obstruct capillaries and restrict blood flow to an organ resultingin ischaemia, pain, necrosis, and often organ damage. The frequency,severity, and duration of these crises vary considerably. Painful crisesare treated with hydration, analgesics, and blood transfusion; painmanagement requires opioid administration at regular intervals until thecrisis has settled. For milder crises, a subgroup of patients manage onnonsteroidal anti-inflammatory drugs (NSAIDs) such as diclofenac ornaproxen. For more severe crises, most patients require inpatientmanagement for intravenous opioids; patient-controlled analgesia devicesare commonly used in this setting. Vaso-occlusive crisis involvingorgans such as the penis or lungs are considered an emergency andtreated with red-blood cell transfusions. Incentive spirometry, atechnique to encourage deep breathing to minimize the development ofatelectasis, is recommended.

Because of its narrow vessels and function in clearing defective redblood cells, the spleen is frequently affected. It is usually infarctedbefore the end of childhood in individuals suffering from sickle-cellanemia. This spleen damage increases the risk of infection fromencapsulated organisms; preventive antibiotics and vaccinations arerecommended for those lacking proper spleen function.

Splenic sequestration crises are acute, painful enlargements of thespleen, caused by intrasplenic trapping of red cells and resulting in aprecipitous fall in hemoglobin levels with the potential for hypovolemicshock. Sequestration crises are considered an emergency. If not treated,patients may die within 1-2 hours due to circulatory failure. Managementis supportive, sometimes with blood transfusion. These crises aretransient, they continue for 3-4 hours and may last for one day.

Acute chest syndrome (ACS) is defined by at least two of the followingsigns or symptoms: chest pain, fever, pulmonary infiltrate or focalabnormality, respiratory symptoms, or hypoxemia. It is the second-mostcommon complication and it accounts for about 25% of deaths in patientswith SCD, majority of cases present with vaso-occlusive crises then theydevelop ACS. Nevertheless, about 80% of patients have vaso-occlusivecrises during ACS.

Aplastic crises are acute worsenings of the patient's baseline anaemia,producing pale appearance, fast heart rate, and fatigue. This crisis isnormally triggered by parvovirus B19, which directly affects productionof red blood cells by invading the red cell precursors and multiplyingin and destroying them. Parvovirus infection almost completely preventsred blood cell production for two to three days. In normal individuals,this is of little consequence, but the shortened red cell life of SCDpatients' results in an abrupt, life-threatening situation. Reticulocytecounts drop dramatically during the disease (causing reticulocytopenia),and the rapid turnover of red cells leads to the drop in hemoglobin.This crisis takes 4 days to one week to disappear. Most patients can bemanaged supportively; some need blood transfusion.

Haemolytic crises are acute accelerated drops in hemoglobin level. Thered blood cells break down at a faster rate. This is particularly commonin patients with coexistent G6PD deficiency. Management is supportive,sometimes with blood transfusions.

One of the earliest clinical manifestations is dactylitis, presenting asearly as six months of age, and may occur in children with sickle-celltrait. The crisis can last up to a month. Another recognised type ofsickle crisis, acute chest syndrome, is characterized by fever, chestpain, difficulty breathing, and pulmonary infiltrate on a chest X-ray.Given that pneumonia and sickling in the lung can both produce thesesymptoms, the patient is treated for both conditions. It can betriggered by painful crisis, respiratory infection, bone-marrowembolisation, or possibly by atelectasis, opiate administration, orsurgery. Hematopoietic ulcers may also occur.

Normally, humans have hemoglobin A, which consists of two alpha and twobeta chains, hemoglobin A2, which consists of two alpha and two deltachains, and hemoglobin F, consisting of two alpha and two gamma chainsin their bodies. Out of these three types, hemoglobin F dominates untilabout 6 weeks of age. Afterwards, hemoglobin A dominates throughoutlife. In people diagnosed with sickle cell disease, at least one of theβ-globin subunits in hemoglobin A is replaced with what's known ashemoglobin S. In sickle cell anemia, a common form of sickle celldisease, hemoglobin S replaces both β-globin subunits in the hemoglobin.

Sickle-cell conditions have an autosomal recessive pattern ofinheritance from parents. The types of hemoglobin a person makes in thered blood cells depend on what hemoglobin genes are inherited from heror his parents. If one parent has sickle-cell anaemia and the other hassickle-cell trait, then the child has a 50% chance of having sickle-celldisease and a 50% chance of having sickle-cell trait. When both parentshave sickle-cell trait, a child has a 25% chance of sickle-cell disease,25% do not carry any sickle-cell alleles, and 50% have the heterozygouscondition.

Sickle-cell gene mutation probably arose spontaneously in differentgeographic areas, as suggested by restriction endonuclease analysis.These variants are known as Cameroon, Senegal, Benin, Bantu, andSaudi-Asian. Their clinical importance is because some are associatedwith higher HbF levels, e.g., Senegal and Saudi-Asian variants, and tendto have milder disease.

In people heterozygous for HgbS (carriers of sickling hemoglobin), thepolymerisation problems are minor, because the normal allele is able toproduce over 50% of the hemoglobin. In people homozygous for HgbS, thepresence of long-chain polymers of HbS distort the shape of the redblood cell from a smooth doughnut-like shape to ragged and full ofspikes, making it fragile and susceptible to breaking withincapillaries. Carriers have symptoms only if they are deprived of oxygen(for example, while climbing a mountain) or while severely dehydrated.The sickle-cell disease occurs when the sixth amino acid, glutamic acid,is replaced by valine to change its structure and function; as such,sickle-cell anemia is also known as E6V. Valine is hydrophobic, causingthe hemoglobin to collapse on itself occasionally. The structure is notchanged otherwise. When enough hemoglobin collapses on itself the redblood cells become sickle-shaped.

The gene defect is a known mutation of a single nucleotide (seesingle-nucleotide polymorphism—SNP) (A to T) of the β-globin gene, whichresults in glutamic acid (E/Glu) being substituted by valine (V/Val) atposition 6. Hemoglobin S with this mutation is referred to as HbS, asopposed to the normal adult HbA. This is normally a benign mutation,causing no apparent effects on the secondary, tertiary, or quaternarystructures of hemoglobin in conditions of normal oxygen concentration.What it does allow for, under conditions of low oxygen concentration, isthe polymerization of the HbS itself. The deoxy form of hemoglobinexposes a hydrophobic patch on the protein between the E and F helices.The hydrophobic side chain of the valine residue at position 6 of thebeta chain in hemoglobin is able to associate with the hydrophobicpatch, causing HbS molecules to aggregate and form fibrous precipitates.

The allele responsible for sickle-cell anaemia can be found on the shortarm of chromosome 11, more specifically 11p15.5. A person who receivesthe defective gene from both father and mother develops the disease; aperson who receives one defective and one healthy allele remainshealthy, but can pass on the disease and is known as a carrier orheterozygote. Heterozygotes are still able to contract malaria, buttheir symptoms are generally less severe.

Due to the adaptive advantage of the heterozygote, the disease is stillprevalent, especially among people with recent ancestry inmalaria-stricken areas, such as Africa, the Mediterranean, India, andthe Middle East. Malaria was historically endemic to southern Europe,but it was declared eradicated in the mid-20th century, with theexception of rare sporadic cases.

The malaria parasite has a complex lifecycle and spends part of it inred blood cells. In a carrier, the presence of the malaria parasitecauses the red blood cells with defective hemoglobin to ruptureprematurely, making the Plasmodium parasite unable to reproduce.Further, the polymerization of Hb affects the ability of the parasite todigest Hb in the first place. Therefore, in areas where malaria is aproblem, people's chances of survival actually increase if they carrysickle-cell trait (selection for the heterozygote).

In the United States, with no endemic malaria, the prevalence ofsickle-cell anaemia among African Americans is lower (about 0.25%) thanin West Africa (about 4.0%) and is falling. Without endemic malaria, thesickle-cell mutation is purely disadvantageous and tends to decline inthe affected population by natural selection, and now artificiallythrough prenatal genetic screening. However, the African Americancommunity descends from a significant admixture of several African andnon-African ethnic groups and also represents the descendants ofsurvivors of slavery and the slave trade. Thus, a lower degree ofendogamy and, particularly, abnormally high health-selective pressurethrough slavery may be the most plausible explanations for the lowerprevalence of sickle-cell anemia (and, possibly, other genetic diseases)among African Americans compared to West Africans. Another factor thatlimits the spread of sickle-cell genes in North America is the absenceof cultural proclivities to polygamy, which allows affected males tocontinue to seek unaffected children with multiple partners.

B. Transfusion Therapy and Alloimmunization

RBC transfusion is an essential treatment for patients with SCD butdevelopment of alloimmunization is a significant complication thataffects 30-50% of those who require chronic transfusion therapy.Patients with SCD are at higher risk of alloimmunization than othertransfused patient populations, often developing multiple alloantibodiesand broadly reactive antibodies that are difficult to identify. Onemajor explanation for high alloimmunization rates in patients with SCDis the disparate distribution of RBC antigens between patients who areprimarily of African ancestry, and blood donors of non-African ethnicbackgrounds (Vichinsky, 1990). The frequency of C, E, K, Jkb, Fya, Leaand S antigens is significantly lower in individuals of African descentcompared to blood donors who are primarily of European descent. Outsideof ABO, the Rh blood group system is the most immunogenic. Sincesensitization to Rh antigens (D, C, c, E, e) and to K (a Kell systemantigen) comprise a majority of the RBC antibodies encountered inpatients with SCD, consensus guidelines recommend provision of C, E,K-matched RBCs to this patient population (Yawn, 2014). Transfusion withRBC units from African American donors with the same ethnic background,who are more likely to have similar blood group antigen profiles, hasalso been suggested to mitigate exposure to foreign antigens that causehigh rates of alloimmunization. Despite this strategy, alloimmunizationhas remained alarmingly high with 58% of chronic and 15% of episodicallytransfused patients immunized (Chou, 2013).

Antibodies directed against the Rh blood group system were the mostcommon antibodies, suggesting that transfused RBCs were not trulyRh-matched (D, C, and E). Genetic analysis of the two genes, RHD andRHCE, that encode the Rh antigens revealed that >85% of patients carryvariant alleles that result in loss or alteration of Rh antigenicepitopes. Variations in Rh blood group antigen expression are notdetected by traditional blood bank tests which detect only the principalRh antigens (D,C,c,E,e). Consequently, patients with SCD are at risk ofRh antibody formation when exposed to conventional Rh antigens on donorRBCs (Chou, 2013). This problem is unique to patients with SCD who areprimarily of African Black ethnicity, as RH variation occurs in <3% ofindividuals from other ethnic populations, in contrast to 85% of Blacks.In addition to the inability to detect Rh variation on the RBCs ofpatients with SCD, the accurate identification of the Rh antibodies theyform as a consequence of having inherited altered Rh proteins is notpossible with commercial RBC reagents. These RBC reagents are fromCaucasian donors and represent Caucasian RBC phenotypes. This limits theability for blood banks to determine donor compatibility, requirescostly sample referral to specialized reference laboratories, andultimately, delays transfusion and patient care, emphasizing the needfor specialized typing reagents for this patient population.

The Rh system is the most complex of all blood group systems andincludes greater than 50 different antigens encompassing polymorphicepitopes, but “Rh typing” of RBCs tests for five (D,C,c,E,e) common inall populations. The Rh proteins are encoded by two genes: RHD encodesthe D antigen and RHCE encodes the CE antigens in various combinations(ce, cE, Ce, CE). Individuals with D antigen expressed are “Rh positive”and the absence of D, usually due to RHD gene deletion, are “Rhnegative.” The highest incidence of “Rh negative” (15-17%) occurs inCaucasians of European ancestry. The RHD and RHCE genes are inherited asa haplotype with allele frequencies that differ in various populations.

RHD and RHCE genetic variants are frequent in individuals of AfricanBlack ethnicity and result in altered epitopes often termed “partial” Rhantigens because they lack common epitopes. Patients with variant RH wholack commonly encoded epitopes are at risk of antibody production ifexposed to foreign Rh epitopes via transfusion or pregnancy. Thus, RhD+individuals with “partial D” antigen may form anti-D (to the epitopes ofD they lack) when exposed to conventional D antigen (Wagner, 2002;Westhoff, 2010; Chou, 2013). For example, RHD*DAU4 encodes a protein inwhich lysine replaces glutamic acid at amino acid position 233 resultingin loss or alteration of one or more common RhD epitopes. Variant RHCEalleles encoding “partial C, c, or e antigens” occurs frequently inAfrican Blacks, and their RBCs often lack high prevalence Rh antigenicepitopes, such as hrB and hrS, and express novel antigenic epitopes (V,VS) (Noizat-Pirenne, 2011; Denomme, 2014). For example, the relativelycommon altered allele, RHCE*ce(733G), encodes a new antigen VS and lossof the high prevalence antigen hrB. The inventors demonstrated thatvariant RHD or RHCE contributes to Rh alloimmunization and delayedtransfusion reactions in patients with SCD (Chou, 2013). Genotyping canbe used to type patients to identify RH variants and guide antibodyevaluations, but at the hospital level, routine blood bank reagents areneeded to identify anti-Rh antibodies with the precision or “finespecificity” needed for clinical decision-making. While genetic matchingof blood may be feasible in the future, the cost and infrastructure todo so is currently prohibitive. The required high-resolution genotypingof RH and other blood group antigens to truly match patients with SCDcost ˜$2,000/donor unit. With a large exchange transfusion program atCHOP, the average number of units is 7 per transfusion visit. Thus,generating iPSC-derived RBC reagents now to improve antibodyidentification, donor selection, and transfusion safety is critical.

Other antibodies that are often found in the serum of patients with SCDinclude anti-U, anti-hrS, and anti-hrB, but these are heterogeneousspecificities and often are not compatible within each group. RHgenotyping can accurately classify the fine specificity and is key tofinding compatible donors. Approximately 2% (U⁻) to 4% (hrS⁻ and hrB⁻)of African-Americans lack these antigens on their RBCs, and aretherefore at risk of making allo-antibodies. These allo-antibodies,which are clinically significant, cannot be distinguished fromauto-antibodies in traditional testing, as they react with all red cellsprovided with current commercial panels.

It is generally accepted that patients with SCD should have extended RBCtesting performed early in life for the principal clinically significantantigens including Rh (D, C, c, E, e), Kell (K, k), Duffy (Fya, Fyb),Kidd (Jka, Jkb), Dombrock (Doa,Dob), and MNS (M, N, S, s) antigens,which guides donor RBC selection and antibody evaluations. Antibodydetection is performed prior to each transfusion to determine thepresence of antibodies in patient serum directed to RBC antigens.Antibody detection involves incubating the patient's serum against“screening” RBCs which are prepared from group O donors that express themajor antigens in different combinations (FIG. 1 ). Commercial RBCreagents represent common Caucasian RBC antigen phenotypes and do notinclude the “partial” Rh antigens expressed in individuals of AfricanBlack ethnicity.

Two or three reagent RBCs with known antigen profiles are used forroutine antibody detection (FIG. 1 , cells I, II, III). A controlconsisting of the patient's own cells and serum is included to controlfor spontaneous agglutination and to test for the presence of anautoantibody. The patient serum is incubated with reagent RBCs and anantigen-antibody reaction is detected as agglutination of cells,indicating a positive test for antibodies to RBC antigens (FIG. 2 ). Forexample, a patient with anti-K in the serum will show no reactivity withcell I and II, but will be positive with cell III (FIG. 1 ). Whenpositive reactivity is detected in this initial “antibody screening”,the patient's serum is then tested against a panel of 10-12 RBCs withdifferent antigen profiles to definitively determine antibodyspecificity to the blood group antigen. Importantly, antibodyspecificity is determined if the patient's serum reacts with RBCsexpressing the offending antigen and does not react with RBCs lackingthe antigen. Transfusion of RBCs expressing an antigen that the patienthas been immunized against can cause a life-threatening hemolytictransfusion reaction. Thus, it is imperative that the patient receiveRBCs lacking all antigens she/he has been immunized to in theirlifetime.

Positive reactivity with all panel RBCs or “panagglutination” presents adilemma. In FIG. 1 , the patient's serum showed 2-3+ agglutination withthe screening cells I, II, III and the autocontrol. An additional panelof 10-12 cells will also show positive reactions. These results are verydifficult to interpret in chronically transfused patients. The positiveautocontrol suggests the patient has made an antibody to their own RBCs,but this “apparent autoantibody” can be due to an alloantibody bound tocirculating donor cells. This pattern of “panagglutination” results whenthe patient makes an antibody to a high prevalence antigen that isabsent on their RBCs. When this occurs, antibody and compatibilitytesting with donor RBCs becomes complicated and hospital blood banklaboratories must send the samples to reference laboratories.Specialized testing is time-consuming, increases cost, and delaystreatment. To streamline this process, FIG. 1 shows three additionaltyping reagent RBCs that the inventors propose to use for more accurateidentification of antibody specificity in blood banks and improvepatient transfusion therapy.

C. Other Therapeutic Methods

A recombinant red blood cell herein can be administered to a subject inneed thereof to treat the subject. For example, a recombinant red bloodcell described herein can be administered to a patient identified asbeing in need thereof and that is blood antigen compatible for therecombinant red blood cell (e.g., using any of the methods describedherein), with one example being patients with SCD. In other methods, anyrecombinant red blood cell described herein can be administered to apatient that in need thereof (e.g., a patient having a hematologicaldisorder, e.g., hereditary anemia, β-thalassemia, or a hematologiccancer), once the subject has been determined to be compatible for therecombinant red blood cell.

As used herein, “administering”, “transfusing” or “treating” includesreducing the number, frequency, or severity of one or more (e.g., two,three, four, or five) signs or symptoms of a hereditary anemia,β-thalassemia, sickle cell disorder, or cancer in a patient (e.g., anyof the cancers described herein). In some embodiments, administering caninclude providing a blood-compatible tissue and/or blood-compatibleblood product to a patient that is identified as being compatible withthe selected tissue and/or blood product. In some embodiments,administering can delay or inhibit disease progression. In someembodiments, administering is used for massive bleeding transfusion(e.g., emergency massive bleeding transfusion) (e.g., transfusion withO⁻ Rh⁻ null reagent red blood cells).

Non-limiting examples of cancer include acute lymphoblastic leukemia(ALL), acute myeloid leukemia (AML), adrenocortical carcinoma, analcancer, appendix cancer, astrocytoma, basal cell carcinoma, brain tumor,bile duct cancer, bladder cancer, bone cancer, breast cancer, bronchialtumor, Burkitt Lymphoma, carcinoma of unknown primary origin, cardiactumor, cervical cancer, chordoma, chronic lymphocytic leukemia (CLL),chronic myelogenous leukemia (CML), chronic myeloproliferative neoplasm,colon cancer, colorectal cancer, craniopharyngioma, cutaneous T-celllymphoma, ductal carcinoma, embryonal tumor, endometrial cancer,ependymoma, esophageal cancer, esthesioneuroblastoma, fibroushistiocytoma, Ewing sarcoma, eye cancer, germ cell tumor, gallbladdercancer, gastric cancer, gastrointestinal carcinoid tumor,gastrointestinal stromal tumor, gestational trophoblastic disease,glioma, head and neck cancer, hairy cell leukemia, hepatocellularcancer, histiocytosis, Hodgkin lymphoma, hypopharyngeal cancer,intraocular melanoma, islet cell tumor, Kaposi sarcoma, kidney cancer,Langerhans cell histiocytosis, laryngeal cancer, leukemia, lip and oralcavity cancer, liver cancer, lobular carcinoma in situ, lung cancer,lymphoma, macroglobulinemia, malignant fibrous histiocytoma, melanoma,Merkel cell carcinoma, mesothelioma, metastatic squamous neck cancerwith occult primary, midline tract carcinoma, involving NUT gene, mouthcancer, multiple endocrine neoplasia syndrome, multiple myeloma, mycosisfungoides, myelodysplastic syndrome, myelodysplastic/myeloproliferativeneoplasm, nasal cavity and para-nasal sinus cancer, nasopharyngealcancer, neuroblastoma, non-Hodgkin lymphoma, non-small cell lung cancer,oropharyngeal cancer, osteosarcoma, ovarian cancer, pancreatic cancer,papillomatosis, paraganglioma, parathyroid cancer, penile cancer,pharyngeal cancer, pheochromocytomas, pituitary tumor, pleuropulmonaryblastoma, primary central nervous system lymphoma, prostate cancer,rectal cancer, renal cell cancer, renal pelvis and ureter cancer,retinoblastoma, rhabdoid tumor, salivary gland cancer, Sezary syndrome,skin cancer, small cell lung cancer, small intestine cancer, soft tissuesarcoma, spinal cord tumor, stomach cancer, T-cell lymphoma, teratoidtumor, testicular cancer, throat cancer, thy mom and thymic carcinoma,thyroid cancer, urethral cancer, uterine cancer, vaginal cancer, vulvarcancer, and Wilms' tumor. Additional examples of cancer are known in theart.

In some embodiments of any of the methods described herein, the canceris a hematologic cancer (e.g., leukemia (e.g., acute lymphoblasticleukemia (ALL), acute myeloid leukemia (AML), chronic lymphocyticleukemia (CLL), chronic myelogenous leukemia (CML)), lymphoma (e.g.,Hodgkin's lymphoma or non-Hodgkin's lymphoma), or multiple myeloma).

In some embodiments of any of the methods described herein, the canceris characterized by having a population of cancer cells that expressCD38 or CD47. In some embodiments of any of the methods describedherein, the patient has received at least one dose of an anti-CD38therapy or an anti-CD47 therapy.

In some instances, the immunotherapy (e.g., anti-CD38 therapy oranti-CD47 therapy) causes all red blood cells to agglutinate, therebypreventing the detection of common blood group antigens from beingdetected underneath the pan-agglutination caused by the immunotherapy.

A recombinant red blood cell described herein (e.g., a Lua-b⁻recombinant red blood cell or a CD47 null recombinant red blood cell)can be used as a method of treatment, e.g., upon administration ortransfusion in a patient identified that has received an immunotherapythat causes all red blood cells to agglutinate. Any of the recombinantred blood cells described herein can be administered to a subject inneed thereof, where the recombinant red blood cell has been determinedto be compatible with the subject (e.g., using any of the methodsdescribed herein).

III. EXEMPLARY METHODS OF GENETICALLY MODIFYING CELLS

Methods of genetically modifying cells to express an antigen on itssurface (e.g., any of the exemplary blood group antigens describedherein or known in the art) are known in the art. Non-limiting examplesof methods that can be used to introduce a nucleic acid or an expressionvector into a cell include lipofection, transfection, electroporation,microinjection, calcium phosphate transfection, dendrimer-basedtransfection, cationic polymer transfection, cell squeezing,sonoporation, optical transfection, impalection, hydrodynamic delivery,magnetofection, viral transduction (e.g., adenoviral and lentiviraltransduction), or nanoparticle transfection.

Methods of genetically modifying cells to decrease or prevent theexpression of an antigen on its cell surface (e.g., any of the bloodgroup antigens described herein or known in the art) are known in theart. Non-limiting examples of methods that can be used to decrease orprevent the expression of an antigen on the surface of a cell (e.g., anyof the blood group antigens described herein or known in the art)include introducing a nucleic acid or an expression vector that includesan inhibitory nucleic acid (e.g., a short hairpin RNA, a smallinterfering RNA, or a microRNA) targeting a nucleic acid encoding theantigen into the cell by lipofection, transfection, electroporation,microinjection, calcium phosphate transfection, dendrimer-basedtransfection, cationic polymer transfection, cell squeezing,sonoporation, optical transfection, impalection, hydrodynamic delivery,magnetofection, viral transduction (e.g., adenoviral and lentiviraltransduction), or nanoparticle transfection.

In some instances, the CRISPR/Cas9 system or any other site-specificrecombinase is used to delete a gene encoding a cell surface antigen(e.g., any of the blood group antigens described herein or known in theart) from a cell or to introduce a recombinant gene construct to expressan alternative antigen on the cell. Briefly, a guide RNA is designedthat is complementary to the endogenous gene encoding the cell surfaceantigen (e.g., any of the blood group antigens described herein or knownin the art). The designed guide RNA and Cas9 enzyme are introduced intoa cell and the gene of interest (e.g., a gene encoding a cell surfaceantigen) is deleted by homologous recombination. In other examples, atranscription activator-like effector nuclease (TALEN) or azinc-finger-like enzyme can be used to delete or add a gene of interest(e.g., a gene encoding a cell surface antigen) to a cell.

In some instances, the CRISPR/Cas9 system is used to engineer a Group O,Rh-null cell line as a first step (see, e.g., Table 1.4 and FIG. 22A).For example, to disrupt expression of RHCE, a guide RNA is designed thattarget the first or second exon of the RHCE gene. Clones in which repairby non-homologous-end-joining has resulted in the introduction of a stopcodon are screened and selected. iPSCs are first transduced with aninducible Cas9 and guide RNA vectors and successfully transduced cloneswill be selected using antibiotics. Expression of the Cas9 protein islater induced with doxycycline and clones in which mutations have beenintroduced are screened by PCR. Next, other atypical Rh proteins areintroduced into the Rh-null cell line to generate novel cells not foundin humans to date. In some instances, reagent red blood cells aregenerated using a transgene-free approach.

In some examples, a reagent red blood cells (e.g., a first reagent redblood cell or a second reagent red blood cell) produced in thelaboratory can express the same blood group antigen profiles whencompared to a donor RBCs frozen in liquid nitrogen. FIGS. 22B-Dillustrate exemplary reagent red blood cells differentiated from iPSCs,and expression of RhAG (Rh associated glycoprotein) on the donororiginal red cells and from iPSCs.

A. CRISPR and Nucleases

CRISPRs (clustered regularly interspaced short palindromic repeats) areDNA loci containing short repetitions of base sequences. Each repetitionis followed by short segments of “spacer DNA” from previous exposures toa virus. CRISPRs are found in approximately 40% of sequenced eubacteriagenomes and 90% of sequenced archaea. CRISPRs are often associated withCas genes that code for proteins related to CRISPRs. The CRISPR/Cassystem is a prokaryotic immune system that confers resistance to foreigngenetic elements such as plasmids and phages and provides a form ofacquired immunity. CRISPR spacers recognize and silence these exogenousgenetic elements like RNAi in eukaryotic organisms.

Repeats were first described in 1987 for the bacterium Escherichia coli.In 2000, similar clustered repeats were identified in additionalbacteria and archaea and were termed Short Regularly Spaced Repeats(SRSR). SRSR were renamed CRISPR in 2002. A set of genes, some encodingputative nuclease or helicase proteins, were found to be associated withCRISPR repeats (the cas, or CRISPR-associated genes).

In 2005, three independent researchers showed that CRISPR spacers showedhomology to several phage DNA and extrachromosomal DNA such as plasmids.This was an indication that the CRISPR/cas system could have a role inadaptive immunity in bacteria. Koonin and colleagues proposed thatspacers serve as a template for RNA molecules, analogously to eukaryoticcells that use a system called RNA interference.

In 2007 Barrangou, Horvath (food industry scientists at Danisco) andothers showed that they could alter the resistance of Streptococcusthermophilus to phage attack with spacer DNA. Doudna and Charpentier hadindependently been exploring CRISPR-associated proteins to learn howbacteria deploy spacers in their immune defenses. They jointly studied asimpler CRISPR system that relies on a protein called Cas9. They foundthat bacteria respond to an invading phage by transcribing spacers andpalindromic DNA into a long RNA molecule that the cell then usestracrRNA and Cas9 to cut it into pieces called crRNAs.

CRISPR was first shown to work as a genome engineering/editing tool inhuman cell culture by 2012 It has since been used in a wide range oforganisms including baker's yeast (S. cerevisiae), zebra fish, nematodes(C. elegans), plants, mice, and several other organisms. Additionally,CRISPR has been modified to make programmable transcription factors thatallow scientists to target and activate or silence specific genes.Libraries of tens of thousands of guide RNAs are now available.

The first evidence that CRISPR can reverse disease symptoms in livinganimals was demonstrated in 2014, when MIT researchers cured mice of arare liver disorder. Since 2012, the CRISPR/Cas system has been used forgene editing (silencing, enhancing or changing specific genes) that evenworks in eukaryotes like mice and primates. By inserting a plasmidcontaining cas genes and specifically designed CRISPRs, an organism'sgenome can be cut at any desired location.

CRISPR repeats range in size from 24 to 48 base pairs. They usually showsome dyad symmetry, implying the formation of a secondary structure suchas a hairpin, but are not truly palindromic. Repeats are separated byspacers of similar length. Some CRISPR spacer sequences exactly matchsequences from plasmids and phages, although some spacers match theprokaryote's genome (self-targeting spacers). New spacers can be addedrapidly in response to phage infection.

CRISPR-associated (cas) genes are often associated with CRISPRrepeat-spacer arrays. As of 2013, more than forty different Cas proteinfamilies had been described. Of these protein families, Cas1 appears tobe ubiquitous among different CRISPR/Cas systems. Particularcombinations of cas genes and repeat structures have been used to define8 CRISPR subtypes (E coli, Ypest, Nmeni, Dvulg, Tneap, Hmari, Apern, andMtube), some of which are associated with an additional gene moduleencoding repeat-associated mysterious proteins (RAMPs). More than oneCRISPR subtype may occur in a single genome. The sporadic distributionof the CRISPR/Cas subtypes suggests that the system is subject tohorizontal gene transfer during microbial evolution.

Exogenous DNA is apparently processed by proteins encoded by Cas genesinto small elements (˜30 base pairs in length), which are then somehowinserted into the CRISPR locus near the leader sequence. RNAs from theCRISPR loci are constitutively expressed and are processed by Casproteins to small RNAs composed of individual, exogenously-derivedsequence elements with a flanking repeat sequence. The RNAs guide otherCas proteins to silence exogenous genetic elements at the RNA or DNAlevel. Evidence suggests functional diversity among CRISPR subtypes. TheCse (Cas subtype E coli) proteins (called CasA-E in E. coli) form afunctional complex, Cascade, that processes CRISPR RNA transcripts intospacer-repeat units that Cascade retains. In other prokaryotes, Cas6processes the CRISPR transcripts. Interestingly, CRISPR-based phageinactivation in E. coli requires Cascade and Cas3, but not Cas1 andCas2. The Cmr (Cas RAMP module) proteins found in Pyrococcus furiosusand other prokaryotes form a functional complex with small CRISPR RNAsthat recognizes and cleaves complementary target RNAs. RNA-guided CRISPRenzymes are classified as type V restriction enzymes.

Cas9 is a nuclease, an enzyme specialized for cutting DNA, with twoactive cutting sites, one for each strand of the double helix. The teamdemonstrated that they could disable one or both sites while preservingCas9's ability to home located its target DNA. Jinek et al. (2012)combined tracrRNA and spacer RNA into a “single-guide RNA” moleculethat, mixed with Cas9, could find and cut the correct DNA targets. Jineket al. (2012) proposed that such synthetic guide RNAs might be able tobe used for gene editing.

Cas9 proteins are highly enriched in pathogenic and commensal bacteria.CRISPR/Cas-mediated gene regulation may contribute to the regulation ofendogenous bacterial genes, particularly during bacterial interactionwith eukaryotic hosts. For example, Cas protein Cas9 of Francisellanovicida uses a unique, small, CRISPR/Cas-associated RNA (scaRNA) torepress an endogenous transcript encoding a bacterial lipoprotein thatis critical for F. novicida to dampen host response and promotevirulence. Wang et al. (2015) showed that coinjection of Cas9 mRNA andsgRNAs into the germline (zygotes) generated nice with mutations.Delivery of Cas9 DNA sequences also is contemplated.

See also U.S. Patent Publication 2014/0068797, which is incorporated byreference in its entirety.

Clustered Regularly Interspaced Short Palindromic Repeats fromPrevotella and Francisella 1 or CRISPR/Cpf1 is a DNA-editing technologyanalogous to the CRISPR/Cas9 system. Cpf1 is an RNA-guided endonucleaseof a class II CRISPR/Cas system. This acquired immune mechanism is foundin Prevotella and Francisella bacteria. It prevents genetic damage fromviruses. Cpf1 genes are associated with the CRISPR locus, coding for anendonuclease that use a guide RNA to find and cleave viral DNA. Cpf1 isa smaller and simpler endonuclease than Cas9, overcoming some of theCRISPR/Cas9 system limitations. CRISPR/Cpf1 could have multipleapplications, including treatment of genetic illnesses and degenerativeconditions.

CRISPR/Cpf1 was found by searching a published database of bacterialgenetic sequences for promising bits of DNA. Its identification throughbioinformatics as a CRISPR system protein, its naming, and a hiddenMarkov model (HMM) for its detection were provided in 2012 in a releaseof the TIGRFAMs database of protein families. Cpf1 appears in manybacterial species. The ultimate Cpf1 endonuclease that was developedinto a tool for genome editing was taken from one of the first 16species known to harbor it. Two candidate enzymes from Acidaminococcusand Lachnospiraceae display efficient genome-editing activity in humancells.

A smaller version of Cas9 from the bacterium Staphylococcus aureus is apotential alternative to Cpf1.

The systems CRISPR/Cas are separated into three classes. Class 1 usesseveral Cas proteins together with the CRISPR RNAs (crRNA) to build afunctional endonuclease. Class 2 CRISPR systems use a single Cas proteinwith a crRNA. Cpf1 has been recently identified as a Class II, Type VCRISPR/Cas systems containing a 1,300 amino acid protein.

The Cpf1 locus contains a mixed alpha/beta domain, a RuvC-I followed bya helical region, a RuvC-II and a zinc finger-like domain. The Cpf1protein has a RuvC-like endonuclease domain that is similar to the RuvCdomain of Cas9. Furthermore, Cpf1 does not have a HNH endonucleasedomain, and the N-terminal of Cpf1 does not have the alfa-helicalrecognition lobe of Cas9.

Cpf1 CRISPR-Cas domain architecture shows that Cpf1 is functionallyunique, being classified as Class 2, type V CRISPR system. The Cpf1 lociencode Cas1, Cas2 and Cas4 proteins more similar to types I and III thanfrom type II systems. Database searches suggest the abundance ofCpf1-family proteins in many bacterial species.

Functional Cpf1 doesn't need the tracrRNA, therefore, only crRNA isrequired. This benefits genome editing because Cpf1 is not only smallerthan Cas9, but also it has a smaller sgRNA molecule (proximately half asmany nucleotides as Cas9).

The Cpf1-crRNA complex cleaves target DNA or RNA by identification of aprotospacer adjacent motif 5′-YTN-3′ (where “Y” is a pyrimidine and “N”is any nucleobase) or 5′-TTN-3′, in contrast to the G-rich PAM targetedby Cas9. After identification of PAM, Cpf1 introduces a sticky-end-likeDNA double-stranded break of 4 or 5 nucleotides overhang.

The CRISPR/Cpf1 system consist of a Cpf1 enzyme and a guide RNA thatfinds and positions the complex at the correct spot on the double helixto cleave target DNA. CRISPR/Cpf1 systems activity has three stages:

-   -   Adaptation, during which Cast and Cas2 proteins facilitate the        adaptation of small fragments of DNA into the CRISPR array;    -   Formation of crRNAs: processing of pre-cr-RNAs producing of        mature crRNAs to guide the Cas protein; and    -   Interference, in which the Cpf1 is bound to a crRNA to form a        binary complex to identify and cleave a target DNA sequence.

B. sgRNA

As an RNA guided protein, Cas9 requires a short RNA to direct therecognition of DNA targets (Mali et al., 2013a). Though Cas9preferentially interrogates DNA sequences containing a PAM sequence NGGit can bind here without a protospacer target. However, the Cas9-sgRNAcomplex requires a close match to the sgRNA to create a double strandbreak (Cho et al., 2013; Hsu et al., 2013). CRISPR sequences in bacteriaare expressed in multiple RNAs and then processed to create guidestrands for RNA (Bikard et al., 2013). Because Eukaryotic systems lacksome of the proteins required to process CRISPR RNAs the syntheticconstruct sgRNA was created to combine the essential pieces of RNA forCas9 targeting into a single RNA expressed with the RNA polymerase typeIII promoter U6 (Mali et al., 2013b,c). Synthetic sgRNAs are slightlyover 100 bp at the minimum length and contain a portion which is targetsthe 20 protospacer nucleotides immediately preceding the PAM sequenceNGG; sgRNAs do not contain a PAM sequence.

IV. PLURIPOTENT CELLS

In some instances, the inventors will utilize pluripotent stems cells(PSCs), such as induced PSCs. Pluripotent stem cells are master cells,which are capable of making cells from all three basic body layers. Assuch, they can potentially produce any cell or tissue the body needs torepair itself. This “master” property is called pluripotency.

Pluripotent stem cells hold great promise in the field of regenerativemedicine. Because they can propagate indefinitely, as well as give riseto every other cell type in the body (such as neurons, heart,pancreatic, and liver cells), they represent a single source of cellsthat could be used to replace those lost to damage or disease. The mostwell-known type of pluripotent stem cell is the embryonic stem cell.However, since the generation of embryonic stem cells involvesdestruction (or at least manipulation) of the pre-implantation stageembryo, there has been much controversy surrounding their use. Further,because embryonic stem cells can only be derived from embryos, it has sofar not been feasible to create patient-matched embryonic stem celllines.

Induced pluripotent stem cells (also known as iPS cells or iPSCs) are atype of pluripotent stem cell that can be generated directly from adultcells. Since iPSCs can be derived directly from adult tissues, they notonly bypass the need for embryos, but can be made in a patient-matchedmanner, which means that each individual could have their ownpluripotent stem cell line. These unlimited supplies of autologous cellscould be used to generate transplants without the risk of immunerejection. While the iPSC technology has not yet advanced to a stagewhere therapeutic transplants have been deemed safe, iPSCs are readilybeing used in personalized drug discovery efforts and understanding thepatient-specific basis of disease.

iPSCs are typically derived by introducing products of specific sets ofpluripotency-associated genes, or “reprogramming factors,” into a givencell type. The original set of reprogramming factors (also dubbedYamanaka factors) are the transcription factors Oct4 (Pou5f1), Sox2,cMyc, and Klf4. While this combination is most conventional in producingiPSCs, each of the factors can be functionally replaced by relatedtranscription factors, miRNAs, small molecules, or even non-relatedgenes such as lineage specifiers.

iPSC derivation is typically a slow and inefficient process, taking 1-2weeks for mouse cells and 3-4 weeks for human cells, with efficienciesaround 0.01%-0.1%. However, considerable advances have been made inimproving the efficiency and the time it takes to obtain iPSCs. Uponintroduction of reprogramming factors, cells begin to form colonies thatresemble pluripotent stem cells, which can be isolated based on theirmorphology, conditions that select for their growth, or throughexpression of surface markers or reporter genes.

In 2014, type O red blood cells were synthesized at the ScottishNational Blood Transfusion Service from iPSC. The cells were induced tobecome a mesoderm, then blood cells, and then red blood cells. A finalstep was to make them eject their nuclei and mature properly.

V. NUCLEIC ACID DELIVERY

In cell engineering studies, expression cassettes are employed toexpress a transcription product. Expression requires that appropriatesignals be provided in the vectors, and include various regulatoryelements such as enhancers/promoters from both viral and mammaliansources that drive expression of the genes of interest in cells.Elements designed to optimize messenger RNA stability andtranslatability in host cells also are defined.

A. Regulatory Elements

Throughout this application, the term “expression cassette” is meant toinclude any type of genetic construct containing a nucleic acid codingfor a gene product in which part or all of the nucleic acid encodingsequence is capable of being transcribed and translated, i.e., is underthe control of a promoter. A “promoter” refers to a DNA sequencerecognized by the synthetic machinery of the cell, or introducedsynthetic machinery, required to initiate the specific transcription ofa gene. The phrase “under transcriptional control” means that thepromoter is in the correct location and orientation in relation to thenucleic acid to control RNA polymerase initiation and expression of thegene. An “expression vector” is meant to include expression cassettescomprised in a genetic construct that is capable of replication, andthus including one or more of origins of replication, transcriptiontermination signals, poly-A regions, selectable markers, andmultipurpose cloning sites.

The term promoter will be used here to refer to a group oftranscriptional control modules that are clustered around the initiationsite for RNA polymerase II. Much of the thinking about how promoters areorganized derives from analyses of several viral promoters, includingthose for the HSV thymidine kinase (tk) and SV40 early transcriptionunits. These studies, augmented by more recent work, have shown thatpromoters are composed of discrete functional modules, each consistingof approximately 7-20 bp of DNA, and containing one or more recognitionsites for transcriptional activator or repressor proteins.

At least one module in each promoter functions to position the startsite for RNA synthesis. The best known example of this is the TATA box,but in some promoters lacking a TATA box, such as the promoter for themammalian terminal deoxynucleotidyl transferase gene and the promoterfor the SV40 late genes, a discrete element overlying the start siteitself helps to fix the place of initiation.

Additional promoter elements regulate the frequency of transcriptionalinitiation. Typically, these are located in the region 30-110 bpupstream of the start site, although a number of promoters have recentlybeen shown to contain functional elements downstream of the start siteas well. The spacing between promoter elements frequently is flexible,so that promoter function is preserved when elements are inverted ormoved relative to one another. In the tk promoter, the spacing betweenpromoter elements can be increased to 50 bp apart before activity beginsto decline. Depending on the promoter, it appears that individualelements can function either co-operatively or independently to activatetranscription.

In certain embodiments, viral promotes such as the human cytomegalovirus(CMV) immediate early gene promoter, the SV40 early promoter, the Roussarcoma virus long terminal repeat, rat insulin promoter andglyceraldehyde-3-phosphate dehydrogenase can be used to obtainhigh-level expression of the coding sequence of interest. The use ofother viral or mammalian cellular or bacterial phage promoters which arewell-known in the art to achieve expression of a coding sequence ofinterest is contemplated as well, provided that the levels of expressionare sufficient for a given purpose. By employing a promoter withwell-known properties, the level and pattern of expression of theprotein of interest following transfection or transformation can beoptimized. Further, selection of a promoter that is regulated inresponse to specific physiologic signals can permit inducible expressionof the gene product.

Enhancers are genetic elements that increase transcription from apromoter located at a distant position on the same molecule of DNA.Enhancers are organized much like promoters. That is, they are composedof many individual elements, each of which binds to one or moretranscriptional proteins. The basic distinction between enhancers andpromoters is operational. An enhancer region as a whole must be able tostimulate transcription at a distance; this need not be true of apromoter region or its component elements. On the other hand, a promotermust have one or more elements that direct initiation of RNA synthesisat a particular site and in a particular orientation, whereas enhancerslack these specificities. Promoters and enhancers are often overlappingand contiguous, often seeming to have a very similar modularorganization.

Below is a list of promoters/enhancers and inducible promoters/enhancersthat could be used in combination with the nucleic acid encoding a geneof interest in an expression construct. Additionally, anypromoter/enhancer combination (as per the Eukaryotic Promoter Data BaseEPDB) could also be used to drive expression of the gene. Eukaryoticcells can support cytoplasmic transcription from certain bacterialpromoters if the appropriate bacterial polymerase is provided, either aspart of the delivery complex or as an additional genetic expressionconstruct.

Where a cDNA insert is employed, one will typically desire to include apolyadenylation signal to effect proper polyadenylation of the genetranscript. The nature of the polyadenylation signal is not believed tobe crucial to the successful practice of the invention, and any suchsequence may be employed such as human growth hormone and SV40polyadenylation signals. Also contemplated as an element of theexpression cassette is a terminator. These elements can serve to enhancemessage levels and to minimize read through from the cassette into othersequences.

B. 2A Protease

The 2A-like self-cleaving domain is derived from the insect virus Thoseaasigna (TaV 2A peptide) (Chang et al., 2009). 2A-like domains have beenshown to function across eukaryotes and cause cleavage of amino acids tooccur co-translationally within the 2A-like peptide domain. Therefore,inclusion of TaV 2A peptide allows the expression of multiple proteinsfrom a single mRNA transcript. Importantly, the domain of TaV whentested in eukaryotic systems have shown greater than 99% cleavageactivity (Donnelly et al., 2001).

C. Delivery of Expression Vectors

There are a number of ways in which expression vectors may introducedinto cells. In certain embodiments of the invention, the expressionconstruct comprises a virus or engineered construct derived from a viralgenome. The ability of certain viruses to enter cells viareceptor-mediated endocytosis, to integrate into host cell genome andexpress viral genes stably and efficiently have made them attractivecandidates for the transfer of foreign genes into mammalian cells(Ridgeway, 1988; Nicolas and Rubenstein, 1988; Baichwal and Sugden,1986; Temin, 1986). The first viruses used as gene vectors were DNAviruses including the papovaviruses (simian virus 40, bovine papillomavirus, and polyoma) (Ridgeway, 1988; Baichwal and Sugden, 1986) andadenoviruses (Ridgeway, 1988; Baichwal and Sugden, 1986). These have arelatively low capacity for foreign DNA sequences and have a restrictedhost spectrum. Furthermore, their oncogenic potential and cytopathiceffects in permissive cells raise safety concerns. They can accommodateonly up to 8 kB of foreign genetic material but can be readilyintroduced in a variety of cell lines and laboratory animals (Nicolasand Rubenstein, 1988; Temin, 1986).

One particular method for in vivo delivery involves the use of anadenovirus expression vector. “Adenovirus expression vector” is meant toinclude those constructs containing adenovirus sequences sufficient to(a) support packaging of the construct and (b) to express an antisensepolynucleotide that has been cloned therein. In this context, expressiondoes not require that the gene product be synthesized.

The expression vector comprises a genetically engineered form ofadenovirus. Knowledge of the genetic organization of adenovirus, a 36kB, linear, double-stranded DNA virus, allows substitution of largepieces of adenoviral DNA with foreign sequences up to 7 kB (Grunhaus andHorwitz, 1992). In contrast to retrovirus, the adenoviral infection ofhost cells does not result in chromosomal integration because adenoviralDNA can replicate in an episomal manner without potential genotoxicity.Also, adenoviruses are structurally stable, and no genome rearrangementhas been detected after extensive amplification. Adenovirus can infectvirtually all epithelial cells regardless of their cell cycle stage. Sofar, adenoviral infection appears to be linked only to mild disease suchas acute respiratory disease in humans.

Adenovirus is particularly suitable for use as a gene transfer vectorbecause of its mid-sized genome, ease of manipulation, high titer, widetarget cell range and high infectivity. Both ends of the viral genomecontain 100-200 base pair inverted repeats (ITRs), which are ciselements necessary for viral DNA replication and packaging. The early(E) and late (L) regions of the genome contain different transcriptionunits that are divided by the onset of viral DNA replication. The E1region (E1A and E1B) encodes proteins responsible for the regulation oftranscription of the viral genome and a few cellular genes. Theexpression of the E2 region (E2A and E2B) results in the synthesis ofthe proteins for viral DNA replication. These proteins are involved inDNA replication, late gene expression and host cell shut-off (Renan,1990). The products of the late genes, including the majority of theviral capsid proteins, are expressed only after significant processingof a single primary transcript issued by the major late promoter (MLP).The MLP, (located at 16.8 m.u.) is particularly efficient during thelate phase of infection, and all the mRNAs issued from this promoterpossess a 5′-tripartite leader (TPL) sequence which makes them mRNAs fortranslation.

In one system, recombinant adenovirus is generated from homologousrecombination between shuttle vector and provirus vector. Due to thepossible recombination between two proviral vectors, wild-typeadenovirus may be generated from this process. Therefore, it is criticalto isolate a single clone of virus from an individual plaque and examineits genomic structure.

Generation and propagation of the current adenovirus vectors, which arereplication deficient, depend on a unique helper cell line, designated293, which was transformed from human embryonic kidney cells by Ad5 DNAfragments and constitutively expresses E1 proteins (Graham et al.,1977). Since the E3 region is dispensable from the adenovirus genome(Jones and Shenk, 1978), the current adenovirus vectors, with the helpof 293 cells, carry foreign DNA in either the E1, the D3 or both regions(Graham and Prevec, 1991). In nature, adenovirus can packageapproximately 105% of the wild-type genome (Ghosh-Choudhury et al.,1987), providing capacity for about 2 extra kb of DNA. Combined with theapproximately 5.5 kb of DNA that is replaceable in the E1 and E3regions, the maximum capacity of the current adenovirus vector is under7.5 kb, or about 15% of the total length of the vector. More than 80% ofthe adenovirus viral genome remains in the vector backbone and is thesource of vector-borne cytotoxicity. Also, the replication deficiency ofthe E1-deleted virus is incomplete.

Helper cell lines may be derived from human cells such as humanembryonic kidney cells, muscle cells, hematopoietic cells or other humanembryonic mesenchymal or epithelial cells. Alternatively, the helpercells may be derived from the cells of other mammalian species that arepermissive for human adenovirus. Such cells include, e.g., Vero cells orother monkey embryonic mesenchymal or epithelial cells. As stated above,the preferred helper cell line is 293.

Racher et al. (1995) disclosed improved methods for culturing 293 cellsand propagating adenovirus. In one format, natural cell aggregates aregrown by inoculating individual cells into 1 liter siliconized spinnerflasks (Techne, Cambridge, UK) containing 100-200 ml of medium.Following stirring at 40 rpm, the cell viability is estimated withtrypan blue. In another format, Fibra-Cel microcarriers (Bibby Sterlin,Stone, UK) (5 g/1) is employed as follows. A cell inoculum, resuspendedin 5 ml of medium, is added to the carrier (50 ml) in a 250 mlErlenmeyer flask and left stationary, with occasional agitation, for 1to 4 h. The medium is then replaced with 50 ml of fresh medium andshaking initiated. For virus production, cells are allowed to grow toabout 80% confluence, after which time the medium is replaced (to 25% ofthe final volume) and adenovirus added at an MOI of 0.05. Cultures areleft stationary overnight, following which the volume is increased to100% and shaking commenced for another 72 h.

Other than the requirement that the adenovirus vector be replicationdefective, or at least conditionally defective, the nature of theadenovirus vector is not believed to be crucial to the successfulpractice of the invention. The adenovirus may be of any of the 42different known serotypes or subgroups A-F. Adenovirus type 5 ofsubgroup C is the preferred starting material in order to obtain theconditional replication-defective adenovirus vector for use in thepresent invention. This is because Adenovirus type 5 is a humanadenovirus about which a great deal of biochemical and geneticinformation is known, and it has historically been used for mostconstructions employing adenovirus as a vector.

As stated above, the typical vector according to the present inventionis replication defective and will not have an adenovirus E1 region.Thus, it will be most convenient to introduce the polynucleotideencoding the gene of interest at the position from which the E1-codingsequences have been removed. However, the position of insertion of theconstruct within the adenovirus sequences is not critical to theinvention. The polynucleotide encoding the gene of interest may also beinserted in lieu of the deleted E3 region in E3 replacement vectors, asdescribed by Karlsson et al. (1986), or in the E4 region where a helpercell line or helper virus complements the E4 defect.

Adenovirus is easy to grow and manipulate and exhibits broad host rangein vitro and in vivo. This group of viruses can be obtained in hightiters, e.g., 10⁹-10¹² plaque-forming units per ml, and they are highlyinfective. The life cycle of adenovirus does not require integrationinto the host cell genome. The foreign genes delivered by adenovirusvectors are episomal and, therefore, have low genotoxicity to hostcells. No side effects have been reported in studies of vaccination withwild-type adenovirus (Couch et al., 1963; Top et al., 1971),demonstrating their safety and therapeutic potential as in vivo genetransfer vectors.

Adenovirus vectors have been used in eukaryotic gene expression (Levreroet al., 1991; Gomez-Foix et al., 1992) and vaccine development (Grunhausand Horwitz, 1992; Graham and Prevec, 1991). Animal studies suggestedthat recombinant adenovirus could be used for gene therapy(Stratford-Perricaudet and Perricaudet, 1991; Stratford-Perricaudet etal., 1990; Rich et al., 1993). Studies in administering recombinantadenovirus to different tissues include trachea instillation (Rosenfeldet al., 1991; Rosenfeld et al., 1992), muscle injection (Ragot et al.,1993), peripheral intravenous injections (Herz and Gerard, 1993) andstereotactic inoculation into the brain (Le Gal La Salle et al., 1993).

The retroviruses are a group of single-stranded RNA virusescharacterized by an ability to convert their RNA to double-stranded DNAin infected cells by a process of reverse-transcription (Coffin, 1990).The resulting DNA then stably integrates into cellular chromosomes as aprovirus and directs synthesis of viral proteins. The integrationresults in the retention of the viral gene sequences in the recipientcell and its descendants. The retroviral genome contains three genes,gag, pol, and env that code for capsid proteins, polymerase enzyme, andenvelope components, respectively. A sequence found upstream from thegag gene contains a signal for packaging of the genome into virions. Twolong terminal repeat (LTR) sequences are present at the 5′ and 3′ endsof the viral genome. These contain strong promoter and enhancersequences and are also required for integration in the host cell genome(Coffin, 1990).

In order to construct a retroviral vector, a nucleic acid encoding agene of interest is inserted into the viral genome in the place ofcertain viral sequences to produce a virus that isreplication-defective. In order to produce virions, a packaging cellline containing the gag, pol, and env genes but without the LTR andpackaging components is constructed (Mann et al., 1983). When arecombinant plasmid containing a cDNA, together with the retroviral LTRand packaging sequences is introduced into this cell line (by calciumphosphate precipitation for example), the packaging sequence allows theRNA transcript of the recombinant plasmid to be packaged into viralparticles, which are then secreted into the culture media (Nicolas andRubenstein, 1988; Temin, 1986; Mann et al., 1983). The media containingthe recombinant retroviruses is then collected, optionally concentrated,and used for gene transfer. Retroviral vectors are able to infect abroad variety of cell types. However, integration and stable expressionrequire the division of host cells (Paskind et al., 1975).

A novel approach designed to allow specific targeting of retrovirusvectors was recently developed based on the chemical modification of aretrovirus by the chemical addition of lactose residues to the viralenvelope. This modification could permit the specific infection ofhepatocytes via sialoglycoprotein receptors.

A different approach to targeting of recombinant retroviruses wasdesigned in which biotinylated antibodies against a retroviral envelopeprotein and against a specific cell receptor were used. The antibodieswere coupled via the biotin components by using streptavidin (Roux etal., 1989). Using antibodies against major histocompatibility complexclass I and class II antigens, they demonstrated the infection of avariety of human cells that bore those surface antigens with anecotropic virus in vitro (Roux et al., 1989).

There are certain limitations to the use of retrovirus vectors in allaspects of the present invention. For example, retrovirus vectorsusually integrate into random sites in the cell genome. This can lead toinsertional mutagenesis through the interruption of host genes orthrough the insertion of viral regulatory sequences that can interferewith the function of flanking genes (Varmus et al., 1981). Anotherconcern with the use of defective retrovirus vectors is the potentialappearance of wild-type replication-competent virus in the packagingcells. This can result from recombination events in which theintact-sequence from the recombinant virus inserts upstream from thegag, pol, env sequence integrated in the host cell genome. However, newpackaging cell lines are now available that should greatly decrease thelikelihood of recombination (Markowitz et al., 1988; Hersdorffer et al.,1990).

Other viral vectors may be employed as expression constructs in thepresent invention. Vectors derived from viruses such as vaccinia virus(Ridgeway, 1988; Baichwal and Sugden, 1986; Coupar et al., 1988)adeno-associated virus (AAV) (Ridgeway, 1988; Baichwal and Sugden, 1986;Hermonat and Muzycska, 1984) and herpesviruses may be employed. Theyoffer several attractive features for various mammalian cells(Friedmann, 1989; Ridgeway, 1988; Baichwal and Sugden, 1986; Coupar etal., 1988; Horwich et al., 1990).

In order to effect expression of sense or antisense gene constructs, theexpression construct must be delivered into a cell. This delivery may beaccomplished in vitro, as in laboratory procedures for transformingcells lines, or in vivo or ex vivo, as in the treatment of certaindisease states. One mechanism for delivery is via viral infection wherethe expression construct is encapsidated in an infectious viralparticle.

Several non-viral methods for the transfer of expression constructs intocultured mammalian cells also are contemplated by the present invention.These include calcium phosphate precipitation (Graham and Van Der Eb,1973; Chen and Okayama, 1987; Rippe et al., 1990) DEAE-dextran (Gopal,1985), electroporation (Tur-Kaspa et al., 1986; Potter et al., 1984),direct microinjection (Harland and Weintraub, 1985), DNA-loadedliposomes (Nicolau and Sene, 1982; Fraley et al., 1979) andlipofectamine-DNA complexes, cell sonication (Fechheimer et al., 1987),gene bombardment using high velocity microprojectiles (Yang et al.,1990), and receptor-mediated transfection (Wu and Wu, 1987; Wu and Wu,1988). Some of these techniques may be successfully adapted for in vivoor ex vivo use.

Once the expression construct has been delivered into the cell thenucleic acid encoding the gene of interest may be positioned andexpressed at different sites. In certain embodiments, the nucleic acidencoding the gene may be stably integrated into the genome of the cell.This integration may be in the cognate location and orientation viahomologous recombination (gene replacement) or it may be integrated in arandom, non-specific location (gene augmentation). In yet furtherembodiments, the nucleic acid may be stably maintained in the cell as aseparate, episomal segment of DNA. Such nucleic acid segments or“episomes” encode sequences sufficient to permit maintenance andreplication independent of or in synchronization with the host cellcycle. How the expression construct is delivered to a cell and where inthe cell the nucleic acid remains is dependent on the type of expressionconstruct employed.

In yet another embodiment of the invention, the expression construct maysimply consist of naked recombinant DNA or plasmids. Transfer of theconstruct may be performed by any of the methods mentioned above whichphysically or chemically permeabilize the cell membrane. This isparticularly applicable for transfer in vitro but it may be applied toin vivo use as well. also demonstrated that direct intraperitonealinjection of calcium phosphate-precipitated plasmids results inexpression of the transfected genes. It is envisioned that DNA encodinga gene of interest may also be transferred in a similar manner in vivoand express the gene product.

In still another embodiment of the invention for transferring a nakedDNA expression construct into cells may involve particle bombardment.This method depends on the ability to accelerate DNA-coatedmicroprojectiles to a high velocity allowing them to pierce cellmembranes and enter cells without killing them (Klein et al., 1987).Several devices for accelerating small particles have been developed.One such device relies on a high voltage discharge to generate anelectrical current, which in turn provides the motive force (Yang etal., 1990). The microprojectiles used have consisted of biologicallyinert substances such as tungsten or gold beads.

Selected organs including the liver, skin, and muscle tissue of rats andmice have been bombarded in vivo (Yang et al., 1990; Zelenin et al.,1991). This may require surgical exposure of the tissue or cells, toeliminate any intervening tissue between the gun and the target organ,i.e., ex vivo treatment. Again, DNA encoding a particular gene may bedelivered via this method and still be incorporated by the presentinvention.

In a further embodiment of the invention, the expression construct maybe entrapped in a liposome. Liposomes are vesicular structurescharacterized by a phospholipid bilayer membrane and an inner aqueousmedium. Multilamellar liposomes have multiple lipid layers separated byaqueous medium. They form spontaneously when phospholipids are suspendedin an excess of aqueous solution. The lipid components undergoself-rearrangement before the formation of closed structures and entrapwater and dissolved solutes between the lipid bilayers (Ghosh andBachhawat, 1991). Also contemplated are lipofectamine-DNA complexes.

Liposome-mediated nucleic acid delivery and expression of foreign DNA invitro has been very successful. Wong et al., (1980) demonstrated thefeasibility of liposome-mediated delivery and expression of foreign DNAin cultured chick embryo, HeLa and hepatoma cells. Nicolau et al.,(1987) accomplished successful liposome-mediated gene transfer in ratsafter intravenous injection. A reagent known as Lipofectamine 2000™ iswidely used and commercially available.

In certain embodiments of the invention, the liposome may be complexedwith a hemagglutinating virus (HVJ). This has been shown to facilitatefusion with the cell membrane and promote cell entry ofliposome-encapsulated DNA (Kaneda et al., 1989). In other embodiments,the liposome may be complexed or employed in conjunction with nuclearnon-histone chromosomal proteins (HMG-1) (Kato et al., 1991). In yetfurther embodiments, the liposome may be complexed or employed inconjunction with both HVJ and HMG-1. In that such expression constructshave been successfully employed in transfer and expression of nucleicacid in vitro and in vivo, then they are applicable for the presentinvention. Where a bacterial promoter is employed in the DNA construct,it also will be desirable to include within the liposome an appropriatebacterial polymerase.

Other expression constructs which can be employed to deliver a nucleicacid encoding a particular gene into cells are receptor-mediateddelivery vehicles. These take advantage of the selective uptake ofmacromolecules by receptor-mediated endocytosis in almost all eukaryoticcells. Because of the cell type-specific distribution of variousreceptors, the delivery can be highly specific (Wu and Wu, 1993).

Receptor-mediated gene targeting vehicles generally consist of twocomponents: a cell receptor-specific ligand and a DNA-binding agent.Several ligands have been used for receptor-mediated gene transfer. Themost extensively characterized ligands are asialoorosomucoid (ASOR) (Wuand Wu, 1987) and transferrin (Wagner et al., 1990). A syntheticneoglycoprotein, which recognizes the same receptor as ASOR, has beenused as a gene delivery vehicle (Ferkol et al., 1993; Perales et al.,1994) and epidermal growth factor (EGF) has also been used to delivergenes to squamous carcinoma cells (Myers, EP 0273085).

VI. ASSAYS, KITS AND COMPOSITIONS

A. Methods of Determining Blood Group Antigen Compatibility

Reagent red blood cells generated in culture in the laboratory can beused in blood bank standard assays for identifying antibodies to bloodgroup antigens. In some instances, the methods provided herein canreplace current methods of determining blood compatibility. In otherinstances, the methods provided herein can be used in addition tocurrent methods of determining blood compatibility. The method ofdetermining described herein may replace sending patient samples thatappear incompatible by standard methods from a hospital to a referencehospital for extensive testing.

Provided herein are methods of determining blood group antigencompatibility of a patient sample that include: (a) contacting a firstreagent red blood cell with a patient sample containing a plurality ofantibodies; wherein the first reagent red blood cell is characterized bythe presence of one or more (e.g., two or more, three or more, four ormore, five or more, six or more, seven or more, eight or more, nine ormore, ten or more, fifteen or more, twenty or more, twenty-five or more,thirty or more, thirty-five or more, forty or more, forty-five or more,or fifty or more) blood group antigens (e.g., any of the blood groupantigens described herein or known in the art, in any combination) onits surface; and (b) contacting a second reagent red blood cell with thepatient sample; wherein the second reagent red blood cell ischaracterized by the absence of at least one (e.g., one, two, three,four, five, six, seven, eight, nine, or ten) of the one or more cellsurface antigens on its surface; (c) detecting whether agglutinationoccurs upon contacting the first reagent red blood cell with the patientsample; (d) detecting whether agglutination occurs when contacting thesecond reagent blood cell with the patient sample; and (e) identifyingthat the patient sample is compatible with the at least one of the oneor more cell surface antigens when no agglutination is detected in steps(c) and (d), or identifying that the patient sample is not compatiblewith the at least one of the one or more cell surface antigens whenagglutination is detected in step (c) but is not detected in step (d),where: the first reagent red blood cell and the second reagent red bloodcell have the same surface phenotype, except for the at least one cellsurface antigen. In some embodiments of these methods, the one or moreblood group antigens are selected from the group of: a C antigen, an Eantigen, a c antigen, an e antigen, a U antigen, a S antigen, a santigen, a hrS antigen and a hrB antigen. In some embodiments of thesemethods, the one or more cell surface antigens include a Lua antigen, aLub antigen, and a CD74 antigen.

Some embodiments of these methods further include the use of one or more(e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36,37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54,55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72,73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90,91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106,107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120,121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134,135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 146, 147, 148, 149,150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163,164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177,178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191,192, 193, 194, 195, 196, 197, 198, 199, or 200) additional reagent redblood cells, wherein: each additional reagent red blood cell has asurface phenotype that is characterized, at least in part, by theabsence of one or more cell surface antigens; and each reagent red bloodcell used in the method has a different surface phenotype as compared toall the other reagent red blood cells used in the method. Someembodiments of these methods include the use of two or more, three ormore, four or more, six or more, eight or more, ten or more, twelve ormore, fourteen or more, sixteen or more, eighteen or more, or twenty ormore additional reagent red blood cells.

In some examples of these methods, the patient sample is not compatiblewith the at least one of the one or more cell surface antigens whenagglutination is detected in step (c), but is not detected in step (d).In some examples of these methods, steps (c) and (d) are performed atsubstantially the same time.

In general, any of a variety of assay formats may be used to assessbinding of patient antibodies to red blood cell antigens. Some methodsinclude enzyme linked immunosorbent assay (ELISA), radioimmunoassay(RIA), immunoradiometric assay, fluoroimmunoassay, chemiluminescentassay, bioluminescent assay, and Western blot to mention a few. Inparticular, a competitive assay for the detection and quantitation ofblood antigen antibodies in samples also is provided. The steps ofvarious useful immunodetection methods have been described in thescientific literature, such as, e.g., Doolittle and Ben-Zeev (1999),Gulbis and Galand (1993), De Jager et al. (1993), and Nakamura et al.(1987).

In some examples, the detection of agglutination occurs using opticalspectrometry (e.g., by detecting a change in optical density usingmethods known in the art). In some examples, the first and the secondreagent red blood cell is labelled with a dye, a fluorescent molecule,or a luminescent molecule, which allows for ease in detection ofagglutination (e.g., via light absorbance, fluorescence emission, orlight emission, respectively). In some embodiments, a determination ofagglutination or no agglutination is made, in part, by comparison to acontrol assay with reagents that are known not to agglutinate andreagents that are known to agglutinate.

In some embodiments of any of the methods described herein, the firstreagent red blood cell is characterized, at least in part, by thepresence of a C antigen, an E antigen, a c antigen, and an e antigen onits surface. In some embodiments, the first reagent red blood cell isfurther characterized by the presence of a D antigen on its surface. Insome embodiments, the first reagent red blood cell is furthercharacterized by the absence of a D antigen on its surface.

In some embodiments of any of the methods of determining blood groupantigen compatibility, the second reagent red blood cell ischaracterized, at least in part, by the absence of a U antigen, a Santigen, and a s antigen on its surface. In some embodiments, the secondreagent red blood cell is further characterized by the absence of a Dantigen on its cell surface.

In some embodiments of any of the methods of determining blood groupantigen compatibility, the second reagent red blood cell ischaracterized, at least in part, by the absence of a D antigen on itssurface and the absence of a C antigen, an E antigen, a c antigen, andan e antigen on its surface. In some embodiments, the second reagent redblood cell is further characterized by the presence of a Go(a) antigenon its surface. In some embodiments, the second reagent red blood cellis further characterized by the presence of a DAK antigen on itssurface.

In some embodiments of any of the methods of determining blood groupantigen compatibility, the second reagent red blood cell ischaracterized, at least in part, by the presence of a Doa antigen and aDob antigen on its cell surface.

In some embodiments of any of the methods of determining blood groupantigen compatibility, the second reagent red blood cell ischaracterized, at least in part, by the absence of a hrB antigen on itssurface. In some embodiments, the second reagent red blood cell isfurther characterized by the absence a hrS antigen on its surface. Insome embodiments, the second reagent red blood cell is furthercharacterized by the absence a D antigen on its surface. In someembodiments, the second reagent red blood cell is further characterizedby the presence of a VS antigen on its surface.

In some embodiments of any of the methods of determining blood groupantigen compatibility, the second reagent red blood cell ischaracterized, at least in part, by the absence of a C antigen, an Eantigen, a c antigen, an e antigen, a U antigen, a S antigen, a santigen, a hrS antigen and a hrB antigen on its cell surface.

In some embodiments of any of the methods of determining blood groupantigen compatibility, the second target red blood cell is characterizedby a phenotype selected from the group of: (i) D−, C−, E−, c−, e−; (ii)D+, C−, E−, c−, e−; (iii) D−, U−, S−, s−; (iv) D−, hrB−, VS+; (v) D−,hrB−, hrS−; (vi) D+, C−, E−, c−, e−, Go(a)+; (vii) D+, C−, E−, c−, e−,DAK+; and (viii) D−, Doa−, Dob−.

In some embodiments, the second reagent red blood cell is characterizedby the absence of a Lua antigen and a Lub antigen on its surface. Insome embodiments, the second reagent red blood cell is characterized bythe absence of a CD47 antigen on its surface. In some embodiments of anyof the methods described herein, the sample is obtained from a patientthat has received an anti-CD38 immunotherapy. In some embodiments of anyof the methods described herein, the sample is obtained from a patientthat has received an anti-CD47 immunotherapy. In some embodiments of anyof the methods described herein, the patient has previously received atleast one blood transfusion or at least one dose of a blood product.

In some embodiments, the patient has or has been diagnosed as havinghereditary anemia. In some embodiments, the patient has or has beendiagnosed as having β-thalassemia. In some embodiments, the patient hasor has been diagnosed as having sickle cell disease. In someembodiments, the patient is in need of a blood transfusion oradministration of a blood product.

Methods of determining blood group antigen compatibility are known inthe art. Non-limiting examples of methods of determining blood groupantigen compatibility include: tube agglutination, gel cardagglutination assays, immunologic gold colloid membrane aspiration test(IMAT), revised dot ELISA, microplate technology, column/gelagglutination, solid phase capture assay, Polymerase chain reaction withsequence-specific priming (PCR-SSP), and Matrix-assisted laserdesorption ionization time of flight mass spectrometry (MALDI-TOF MS),and gene sequencing. These methods of determining blood group antigencompatibility can be used in addition to these methods described herein.

As can be appreciated in the art, one or more (e.g., 2 or more, 3 ormore, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more,10 or more, 11 or more, 12 or more, 13 or more, 14 or more, 15 or more,16 or more, 17 or more, 18 or more, 19 or more, 20 or more, 21 or more,22 or more, 23 or more, 24 or more, 25 or more, 26 or more, 27 or more,28 or more, 29 or more, 30 or more, 35 or more, 40 or more, 45 or more,50 or more, 55 or more, 60 or more, 65 or more, 70 or more, 75 or more,80 or more, 85 or more, 90 or more, 95 or more, or 100 or more)additional different reagent red blood cells having varying surfacephenotypes (differing from any of the other reagent red blood cells usedin the method by the presence or absence of one or more surfaceantigens, e.g., any of the blood group antigens described herein orknown in the art) can also be used in any of the methods describedherein. As can be appreciated by those in the art, in any of the methodsdescribed herein, various comparisons of where agglutination is detectedversus where agglutination is not detected can be used to together toprovide information regarding the antigen compatibility of the samplefrom the patient. Such analyses can be performed usingcomputer-implemented software or an algorithm designed to provide theantigen compatibility information for the sample from the patient.

In some embodiments of these methods, the detection of agglutination isperformed using a buffer that includes one or more stabilizing agents(e.g., preservatives, buffers, anti-bacterial agents, anti-fungalagents, or any of the components of any of the specific buffersdescribed herein) or potentiators to enhance reactivity (e.g. albumin,low ionic strength solutions, polyethylene glycol, and various enzymetreatments known to the art. In some embodiments of these methods, theperformance of any of the methods described herein includes the use ofany of the kits described herein.

In some embodiments of these methods, the methods are performed using agel card, a multi-well assay plate, an array, a microplate, a film, atube, a well, a paper matrix, a capillary, a slide, and a chip in whichor onto which the first reagent red blood cell, the second reagent, andoptionally, one or more additional red blood cell are disposed.

In some examples of any of the methods described herein, the patientsample is a plasma sample or a serum sample. In some examples of any ofthe methods described herein, the patient sample comprises plasma orserum.

Some embodiments of these methods can further include recording thedetermined antigen compatibility in the clinical records (e.g., acomputer readable medium) of the patient. Some embodiments can includeperforming the method multiple times on different samples from thepatient over time (e.g., where one or more different samples obtainedfrom the patient are collected at different time points from thepatient).

Some embodiments of these methods further include: selecting a tissue orblood product that is compatible with the at least one of the one ormore cell surface antigens (e.g., blood group antigens) for a patienthaving its patient sample identified as being compatible with the atleast one of the one or more cell surface antigens (e.g., blood groupantigens); or selecting a tissue or blood product that does not includethe at least one of the one or more cell surface antigens (e.g., bloodgroup antigens) for a patient having its patient sample identified asnot being compatible with the at least one of the one or more cellsurface antigens (e.g., blood group antigens). Some embodiments of thesemethods further include: administering the selected tissue or bloodproduct that is compatible with the at least one of the one or more cellsurface antigens (e.g., blood group antigens) to the patient having itspatient sample identified as being compatible with the at least one ofthe one or more cell surface antigens (e.g., blood group antigens); oradministering the selected tissue or blood product that does not includethe at least one of the one or more cell surface antigens (e.g., bloodgroup antigens) to the patient having its patient sample identified asnot being compatible with the at least one of the one or more cellsurface antigens (e.g., blood group antigens).

Some embodiments of any of the methods described herein further include:transfusing a therapeutically effective amount of a second reagent redblood cell to the patient having its patient sample identified as beingcompatible with the at least one of the one or more cell surfaceantigens (e.g., a second reagent red blood cell that has a surfacephenotype selected from the group of: Lua-b− and CD47−).

Some embodiments of any of the methods of determining blood groupcompatibility described herein further include transfusing the patientwith a recombinant red blood cell (e.g., any recombinant red blood celldescribed herein) that is compatible with the patient's identified bloodgroup. In some embodiments, transfusion is a method of treatment of ahematological disorder (e.g., hereditary anemia, sickle cell disorder,β-thalassemia, or a hematologic cancer).

A particularly useful assay for examining blood antibodies is a gel cardassay, such as that already use to perform the ABO, RhD and Kell bloodgroup typing, and the determination of ABO reverse group confirms theABO group. In the field of transfusion medicine, after A and B antigens,the most important blood group antigen is the D antigen from the Rhblood group system. The determination of RhD is defined by the presenceor absence of the D antigen in the red blood cells. The antigen K orKEL1 is the antigen of the Kell system most important from a clinicalpoint of view, as the corresponding antibody is involved in hemolytictransfusion reactions (HTRs) and in hemolytic disease of the newborn(HDN).

The principle of the test is based on a gel technique described 1985 fordetecting red blood cell agglutination reactions. Plastic cards arecomposed of multiple microtubes. Each microtube is made of a chamber,also known as incubator chamber, at the top of a long and narrowmicrotube, referred to as the column. Buffered gel solution containingantibody (unknown or known, such as anti-A, anti-B, anti-AB, anti-D,anti-K) has been prefilled into the microtube of the plastic card. Theagglutination occurs when the red blood cell antigens react with thecorresponding antibodies, present in the gel solution or in the serum orplasma sample (in the case of reverse grouping test). The gel columnacts as a filter that traps agglutinated red blood cells as they passthrough the gel column during the centrifugation of the card. The gelcolumn separates agglutinated red blood cells from non-agglutinated redblood cells based on size. Any agglutinated red blood cells are capturedat the top of or along the gel column, and non-agglutinated red bloodcells reach the bottom of the microtube forming a pellet.

B. Kits

The present disclosure concerns kits for use with the detection methodsdescribed above. As the engineered cells may be used to detectantibodies in blood samples, both the cells and control antibodies maybe included in the kit. The kits will thus comprise, in suitablecontainer means, a plurality of engineered cells expressing rare bloodantigens, or the absence of blood antigens, and optionally one or moreantibodies to confirm functionality of the assay.

In certain embodiments, the cell may be pre-bound to a solid support,such as a column matrix and/or well of a microtiter plate. The reagentsof the kit may take any one of a variety of forms, including detectablelabels that are associated with or linked to a detection agent.Detectable labels that are associated with or attached to a secondarybinding ligand are also contemplated. Exemplary secondary ligands arethose secondary antibodies that have binding affinity for the firstantibody. Other assays will employ agglutination as a read-out forbinding.

The kits may further comprise a suitably aliquoted composition ofpurified blood antigens, whether labeled or unlabeled, as may be used toprepare a standard curve for a detection assay. The kits may containantibody-label conjugates either in fully conjugated form, in the formof intermediates, or as separate moieties to be conjugated by the userof the kit. The components of the kits may be packaged either in aqueousmedia or in lyophilized form.

The container means of the kits will generally include at least onevial, test tube, flask, bottle, syringe or other container means, intowhich the antibody may be placed, or preferably, suitably aliquoted. Thekits of the present disclosure will also typically include a means forcontaining the antibody, antigen, and any other reagent containers inclose confinement for commercial sale. Such containers may includeinjection or blow-molded plastic containers into which the desired vialsare retained.

Also provided herein are kits that include any of the first reagent redblood cells and/or second reagent red blood cells described herein, anyof the compositions described herein, or any of the pharmaceuticalcompositions described herein.

In some embodiments, the kits can include at least one dose of any ofthe compositions (e.g., pharmaceutical compositions) described herein.In some embodiments, the kits can provide a syringe for administeringany of the pharmaceutical compositions described herein.

In some embodiments, the kits can include instructions for performingany of the methods described herein. In some embodiments of any of thekits described herein, the solid support is selected from the groupconsisting of: a gel card, a multi-well assay plate, an array, amicroplate, a film, a tube, a well, a capillary, a paper matrix, a slideand a chip. In some embodiments of any of the kits described herein, thekit can further include glutaraldehyde or another cross-linker tostabilize antigen epitopes.

In some embodiments, the kits provided herein can include: a compositioncomprising a first reagent red blood cell characterized by the presenceof one or more cell surface antigens (e.g., any of the blood groupsurface antigens described herein or known in the art) on its surface;and a composition including a second reagent red blood cellcharacterized by the absence of at least one of the one or more cellsurface antigens on its surface, where the first reagent red blood celland the second reagent red blood cell have the same surface phenotypeexcept for the at least one cell surface antigen.

Some embodiments of these kits further include one or more (e.g., 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40,41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58,59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76,77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94,95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109,110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123,124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137,138, 139, 140, 141, 142, 143, 144, 146, 147, 148, 149, 150, 151, 152,153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166,167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180,181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194,195, 196, 197, 198, 199, or 200) additional reagent red blood cells,wherein: each additional reagent red blood cell has a surface phenotypethat is characterized, at least in part, by the absence of one or morecell surface antigens; and

each reagent red blood cell in the kit has a different surface phenotypeas compared to all the other reagent red blood cells in the kit. Someembodiments of these kits include the use of two or more, three or more,four or more, six or more, eight or more, ten or more, twelve or more,fourteen or more, sixteen or more, eighteen or more, or twenty or moreadditional reagent red blood cells.

Some embodiments of any of the kits provided herein, can furtheroptionally include a solid support (e.g., any of the solid supportsdescribed herein). In some embodiments, the composition including thefirst reagent red blood cell and/or the composition including the secondreagent red blood cell can include one or more of: an antibiotic (e.g.,chloramphenicol, neomycin, and/or gentamycin), Immucor manufacturerdiluent, adenine, adenosine, Alsever's solution, SAGM, and dextrose.

In some embodiments, the kits provided herein can include a firstreagent red blood cell characterized by the presence of one or more cellsurface antigens (e.g., any of the blood group surface antigensdescribed herein or known in the art) on its surface; and a compositionincluding a second reagent red blood cell characterized by the absenceof at least one of the one or more cell surface antigens on its surface,where the first reagent red blood cell and the second reagent red bloodcell have the same surface phenotype except for the at least one cellsurface antigen. Some embodiments of these kits can further include canfurther include a solid support (e.g., any of the solid supportsdescribed herein).

In some embodiments of any of the kits described herein, the one or morecell surface antigens are selected from the group of: a C antigen, an Eantigen, a c antigen, an e antigen, a U antigen, an S antigen, an santigen, an hrB antigen, a Lua antigen, a Lub antigen, and a CD47antigen.

In some examples of any of the kits described herein, the second reagentred blood cell is characterized, at least in part, by the absence of a Cantigen, an E antigen, a c antigen, and an e antigen on its surface. Insome examples of these kits, the second reagent red blood cell isfurther characterized by the absence of a D antigen on its surface. Inother examples of these kits, the second reagent red blood cell isfurther characterized by the presence of a D antigen on its surface. Insome examples of these kits, the second reagent red blood cell ischaracterized by the absence of a U antigen, a S antigen, and a santigen on its surface. In some examples of these kits, the secondreagent red blood cell is further characterized by the absence of a Dantigen on its cell surface.

In some embodiments of any of the kits described herein, the secondreagent red blood cell is characterized, at least in part, by thepresence of a D antigen on its surface and the absence of a C antigen,an E antigen, a c antigen, and an e antigen on its surface. In someembodiments of these kits, the second reagent red blood cell is furthercharacterized by the presence of a Go(a) antigen on its surface. In someembodiments of these kits, the second reagent red blood cell is furthercharacterized by the presence of a DAK antigen on its surface.

In some embodiments of any of the kits described herein, the secondreagent red blood cell is characterized, at least in part, by theabsence of a Doa antigen and a Dob antigen on its surface.

In some embodiments of any of the kits described herein, the secondreagent red blood cell is characterized, at least in part, by theabsence of an hrB antigen on its surface. In some embodiments of thesekits, the second reagent red blood cell is further characterized by theabsence of an hrS antigen on its surface. In some embodiments of thesekits, the second reagent red blood cell is further characterized by theabsence of a D antigen on its cell surface. In some embodiments of thesekits, the second reagent red blood cell is further characterized by thepresence of a VS antigen on its surface.

In some embodiments of any of the kits described herein, the secondreagent red blood cell is characterized, at least in part, by theabsence of a C antigen, an E antigen, a c antigen, an e antigen, a Uantigen, a S antigen, an s antigen, and an hrB antigen on its surface.

In some embodiments of any of the kits described herein, the secondreagent red blood cell is characterized, at least in part, by a surfacephenotype selected from the group of:

(i) D−, C−, E−, c−, e−;

(ii) D+, C−, E−, c−, e−;

(iii) D−, U−, S−, s−;

(iv) D−, hrB−, VS+;

(v) D−, hrB−, hrS−;

(vi) D+, C−, E−, c−, e−, Go(a)+;

(vii) D+, C−, E−, c−, e−, DAK+;

(viii) D−, Doa−, Dob−;

(ix) Lua-b−;

(x) CD47−; and

(xi) any combination of two or more of the surface phenotypes of (i) to(xi).

In some embodiments of any of the kits described herein, the secondreagent red blood cell is characterized by the absence of a C antigen,an E antigen, a c antigen, an e antigen, a U antigen, a S antigen, an santigen, and an hrB antigen on its surface. In some embodiments of anyof the kits described herein, the second reagent red blood cell ischaracterized by the surface phenotype D−, C−, E−, c−, e−, Doa− andDob−. In some embodiments of any of the kits described herein, thesecond reagent red blood cell is characterized by the cell surfacephenotype D−, C−, E−, c−, e−, Lua− and b−.

Some embodiments of any of the kits described herein further include oneor more (e.g., 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7or more, 8 or more, 9 or more, 10 or more, 11 or more, 12 or more, 13 ormore, 14 or more, 15 or more, 16 or more, 17 or more, 18 or more, 19 ormore, 20 or more, 21 or more, 22 or more, 23 or more, 24 or more, 25 ormore, 26 or more, 27 or more, 28 or more, 29 or more, 30 or more, 35 ormore, 40 or more, 45 or more, 50 or more, 55 or more, 60 or more, 65 ormore, 70 or more, 75 or more, 80 or more, 85 or more, 90 or more, 95 ormore, or 100 or more) additional different reagent red blood cellshaving varying surface phenotypes (each differing from any of the otherreagent red blood cells present in the kit by the presence or absence ofone or more surface antigens, e.g., any of the blood group antigensdescribed herein or known in the art).

Some embodiments of any of the kits described herein can further includeone or more reagents useful for performing an agglutination assay. Someembodiments of any of the kits described herein can further include abuffer useful for performing an agglutination assay.

In some embodiments, a kit can further include an algorithm or softwarethat can assist in the determination of blood group antigencompatibility of a patient sample.

C. Compositions

Also provided herein are compositions (e.g., pharmaceuticalcompositions) that include at least one (e.g., at least 2, at least 3,at least 4, at least 5, at least 6, at least 7, at least 8, at least 9,at least 10, at least 15, at least 20, at least 25, at least 30, atleast 35, at least 40, at least 45, or at least 50, at least 55, atleast 60, at least 65, at least 70, at least 75, at least 80, at least85, at least 90, at least 95, at least 100, at least 105, at least 110,at least 115, at least 120, at least 130, at least 135, at least 140, atleast 145, at least 150, at least 155, at least 160, at least 165, atleast 170, at least 175, at least 180, at least 185, at least 190, atleast 195, or at least 200) of any of the reagent red blood cellsdescribed herein (e.g., any of the first reagent red blood cells, any ofthe second reagent red blood cells, and any of the one or moreadditional reagent red blood cells described herein, or any combinationthereof). In some embodiments where the compositions include at leasttwo of any of the reagent red blood cells described herein, each of theat least two reagent red blood cells can have a different surfacephenotype from the rest of the reagent red blood cells in thecomposition.

For example, a composition provided herein can include a reagent redblood cell that is characterized, at least in part, by a surfacephenotype selected from:

(i) D⁻, C⁻, E⁻, c⁻, e⁻;

(ii) D⁺, C⁻, E⁻, c⁻, e⁻;

(iii) D⁻, U⁻, S⁻, s⁻;

(iv) D⁻, hrB⁻, VS⁺,

(v) D⁻, hrB⁻, hrS⁻;

(vi) C⁻, E⁻, c⁻, e⁻;

(vii) D⁺, C⁻, E⁻, c⁻, e⁻, Go(a)⁺;

(viii) D⁺, C⁻, E⁻, c⁻, e⁻, DAK⁺;

(ix) D⁻, Doa⁻, Dob⁻;

(x) Lua⁻ b⁻;

(xi) CD47; and

(xii) any combination of two or more of the surface phenotypes of (i) to(xi).

For example, a combination can include a reagent red blood that has asurface phenotype characterized by D−, C−, E−, c−, e−, Doa− and Dob−. Insome instances, a composition can include a reagent red blood that has asurface phenotype characterized by D−, C−, E−, c−, e−, Lua− and b−. Insome embodiments, a composition including at least one of any of thereagent red blood cells described herein (e.g., any of the first reagentred blood cells or any of the second reagent red blood cells describedherein) can include one or more of: an antibiotic (e.g.,chloramphenicol, neomycin, and/or gentamycin), Immucor manufacturerdiluent, adenine, adenosine, Alsever's solution, SAGM (Saline, Adenine,Glucose, Mannitol) additive solution, and dextrose

In some embodiments, the compositions (e.g., pharmaceuticalcompositions) can be disposed in a sterile vial or tube, or a pre-loadedsyringe.

In some embodiments, the compositions (e.g., pharmaceuticalcompositions) are formulated for different routes of administration(e.g., intravenous). In some embodiments, the compositions (e.g.,pharmaceutical compositions) can include a pharmaceutically acceptablecarrier (e.g., phosphate buffered saline or Ringers Lactate). Single ormultiple administrations of any of the pharmaceutical compositionsdescribed herein can be given to a subject depending on, for example:the dosage and frequency as required and tolerated by the patient. Adosage of the pharmaceutical composition should provide a sufficientquantity of the selected tissue (e.g., any tissue described herein),blood product (e.g., any recombinant blood cell described herein, anyreagent red blood cell described herein (e.g., any of the second reagentred blood cells described herein), or any blood product describedherein) to effectively treat or ameliorate conditions, diseases, orsymptoms.

Also provided herein are methods of treating a subject in need thereof(e.g., a subject having hereditary anemia, β-thalassemia, sickle celldisorder, cancer (e.g., any of the cancers described herein), trauma, ormassive bleeding), that include administering a therapeuticallyeffective amount of at least one of any of the compositions orpharmaceutical compositions provided herein.

The invention is further described in the following examples, which donot limit the scope of the invention described in the claims.

VII. EXAMPLES

The following examples are included to demonstrate embodiments of thedisclosure. It should be appreciated by those of skill in the art thatthe techniques disclosed in the examples which follow representtechniques discovered by the inventor to function well in the practiceof the disclosure, and thus can be considered to constitute modes forits practice. However, those of skill in the art should, in light of thepresent disclosure, appreciate that many changes can be made in thespecific embodiments which are disclosed and still obtain a like orsimilar result without departing from the spirit and scope of thedisclosure.

Example 1

RBC Alloimmunization in Patients with SCD Despite Transfusion from RhMatched Minority Donors.

A major strategy to decrease alloimmunization in SCD is to provide RBCsmatched for C, E, and K antigens. Transfusion with units from AfricanAmerican donors has also been suggested (Vichinsky, 2001), sinceextended RBC antigen profiles are more likely to be similar to patients.The inventors initiated C, E and K matching with transfusion of AfricanAmerican donor units through a “Blue Tag” program with the inventors'regional blood supplier in 1995. Donors self-identify as AfricanAmerican, a blue tag is attached to their donation, and these units arereserved for patients with SCD at two pediatric hospitals inPhiladelphia (FIG. 3A) (Chou, 2012c). This program has substantiallyincreased local African American blood donations and generates asufficient supply of C, E, and K negative RBC units in Philadelphia.

The inventors performed a 15-year retrospective analysis to assessantigen matching for D, C, E, K and transfusion from African Americandonors on alloimmunization rates, antibody specificity, and clinicalsignificance in pediatric and young adult patients with SCD (Chou, 2013featured article in BLOOD). The study included 182 patients who receiveda total of 44,482 RBC units, demonstrating the tremendous resourcesrequired. Surprisingly, the inventors found 58% of 123 chronic and 15%of 59 episodically transfused patients were alloimmunized (median totalRBC units transfused per patient was 230 and 3, respectively).Two-thirds of the antibodies formed were directed against the same Rhblood group antigens that were targeted for prevention (D, E, e, C, c,FIG. 3B).

Notably, the majority of Rh antibodies occurred in patients whose RBCswere phenotypically positive for the corresponding Rh antigen and wouldnot be expected to form antibodies to a “self” antigen. For example,anti-e antibodies were identified in 16 individuals whose RBCs all typede+ (FIG. 3C). Rh specificities were also identified in antigen-negativepatients who received Rh-matched RBCs and had not been transfusedelsewhere (i.e., C-patients formed anti-C antibodies despite transfusionwith C− units, FIG. 3C). Overall, ⅓ of the antibodies were clinicallysignificant with lower hemoglobin or % hemoglobin S levels than theirbaseline pre-transfusion values, suggestive that the patient hemolyzedthe transfused RBCs (FIG. 3D).

High-resolution genotyping of RHD and RHCE revealed 87% of individualshad variant RH alleles that contributed to Rh alloimmunization (FIG. 4). Overall, greater than one-third of RHD and one-half of RHCE allelesdiffered from conventional sequence, and ˜50% of patients had at leastone variant RHD and 1 variant RHCE. While RH variants encode Rh epitopesthat are likely conformational in nature, relatively small changes inpart of the protein can affect the expression of epitopes in other partsof the protein or result in new epitopes (Daniels, 1998). Variant Rhepitopes are complex and D-like epitopes can be expressed on Rhce(Flegel, 2006; Chen, 2006), C-like epitopes are reported on variant RhDand Rhce proteins (Hipsky, 2012; Westhoff, 2012), and E-like antigens onRhCe proteins (Vege, 2012). These findings highlight the limitations ofcurrent Rh typing, Rh matching by serologic methods, and identificationof Rh antibodies with fine specificity. Importantly, these findings haveled to a change of transfusion practice for patients with SCD. Theinventors have implemented DNA-based RBC typing as the primary methodfor extended RBC typing outside ABO and RhD and perform high resolutionRHD and RHCE genotyping for all patients with SCD. Major advantages ofDNA-based RBC typing is the ability to predict expression for greaterthan 30 antigens and identify variants that can guide antibodyevaluations and choice of donor units. Ongoing collaborative effortshave included RBC genotyping in over 900 patients with SCD and ˜600African-American donors (FIG. 4 ) (Chou et al., 2018). While genotypematching of donors to patients is currently cost-prohibitive, it isimperative that methods are established to correctly identify antibodiesduring pre-transfusion testing and prevent the transfusion ofincompatible donor RBCs that can result in an immune-mediated hemolytictransfusion reaction.

Genome Editing in iPSCs.

To generate iPSC-derived RBC typing reagents, the inventors will usegenome editing technologies to modify genes of interest. The CRISPR/Cas9system allows genetic manipulation in human cells with very highefficiency (reviewed in Li, 2014). The inventors have refined thesetechnologies and have confirmed efficient genome editing in human PSCs.As an example, the creation of an RFP reporter line in the ARX locus isshown (FIG. 5A). Drug resistant clones that underwent homologousrecombination were identified with an efficiency of 50% (FIG. 5B and notshown). The inventors have used the CRISPR/Cas9 system to generateinsertion or deletion (indel) mutations at 10 distinct loci in humanPSCs. Briefly, cells are transfected with Cas9/GFP plus the indicatedguide RNA (gRNA). GFP+ cells are sorted 24 hours followed by plating atclonal density. Clones are picked and examined for indel generation atthe gRNA cut site. While variability of gRNAs has been reported (Byrne,2014; Ran, 2013), indel generation success rate is ˜30% (data notshown).

In some instances, multiple amino acid differences will be required togenerate the Rh variant of interest. In these situations, the inventorswill use homology directed repair with a donor DNA vector, guide RNA andCRISPR/Cas9 to generate iPSCs that will express a number of differentRHD proteins including the conventional and variant antigens (FIGS.18A-C). The inventors constructed an insert with RHD cDNA (exons 2-10 ofany given variant), arms of homology, a PGK promoter, a puromycinresistance cassette and LoxP sites. The guide RNA will target intron 1of the RHCE loci of Rh null iPSCs (generated by RHCE inactivation of aD− iPSC), which will allow use of the endogenous promoter and exon 1 ofRHCE alleles (FIGS. 18A-C).

The inventors will also use ZFNs to efficiently introduce transgenesinto the “safe harbor” PPP1R12C (AAVS1) locus of human PSCs(Tiyaboonchai, 2014). An example of this technology is shown in FIG. 6Ain which the Gp1ba promoter was used to drive expression of themegakaryocyte transcription factor FLI-1. The Gp1ba promoter was used todrive expression of the megakaryocyte inducing transcription factorFLI-1 in this example, (FIG. 6B), but a constitutive promoter such asPGK or EF1a will be used in these studies.

Generation of Customized iPSC Lines Using Genome Editing Technologies.

Genome editing technology technologies will be used on human PSCs forthe generation of rare reagent iPSC-derived RBCs for antibodyidentification in patients with SCD. Importantly, the iPSCs generatedhere are those that would be most beneficial and life saving for futuretransfusion. The approach is to utilize existing iPSC lines generated atCHOP which have been successfully differentiated into hematopoieticcells, and use gene editing techniques to generate a panel of renewableiPSCs that lack high prevalence antigens or that express variant Rhantigens, which is specifically relevant to patients with SCD.

Established iPSC Lines.

From the existing panel of wild-type (WT) iPSC lines generated, ABO andRHD genotyping was performed, revealing nine of the lines as Group O,the “universal” donor phenotype (lacking A and B antigens). For thesestudies, the inventors further characterized lines as “Rh positive”(RhD+) expressing the D antigen and “Rh negative” (RhD−) lacking the Dantigen due to RHD gene deletion found in ˜15% of Caucasians (Table1.1). Of importance to remember is that the Rh proteins are encoded bytwo genes: RHD encodes the D antigen and RHCE encodes the CE antigens invarious combinations (ce, cE, Ce, CE). In addition to Rh detection,these iPSC lines were genetically characterized for >30 clinicallysignificant blood group antigens and high prevalence antigens using theHuman Erythrocyte Antigen (HEA) genotyping platform (Casas, 2015) andother laboratory developed methods for those not determined by HEA.Table 1.1 shows the predicted antigen genotypes relevant for thesestudies.

TABLE 1.1 ABO and RHD genotypes of CHOP wild-type iPSC lines. RHD:PCR-multiplex analysis of exons 4, 7, and inactivating pseudogene. ABO:PCR-restriction fragment length polymorphism testing for nucleotidepositions 261 (O1), 467 (A2), 703 (B) and 1096 (B, O2). Extendedphenotype performed on Human Erythrocyte Antigen (HEA) genotypingplatform. Cell of ABO RHD Predicted Predicted extended antigen type byiPSC line Method origin genotype genotype ABO/D type genotype CHOPWT8Lentivirus Peripheral *01/*01 RHD Group O, RhD+ C+ E− c+ e+ K− Jka+ Fya−Fyb+ S− s+ blood U+ Doa− Dob+ CHOPWT9 Sendai Peripheral *01/*01 RHDGroup O, RhD+ C− E+ c+ e− K− Jka+ Jkb+ Fya− Fyb+ S− s+ blood U+ Doa+Dob+ CHOPWT4 Sendai Fibroblast *01/*01 no RHD Group O, RhD− C− E− c+ e+K− Jka− Jkb+ Fya+ Fyb− S− s+ gene U+ Doa+ Dob+ CHOPWT10 SendaiPeripheral *01/*01 no RHD Group O, RhD− C− E− c+ e+ K− Jka− Jkb+ Fya+Fyb− S− s+ blood gene U+ Doa+ Dob+

TABLE 1.2 Rare iPSC-derived RBC reagent panel. Used in combination withexisting RBC panels, these iPSC-derived RBC reagents would facilitaterapid identification of alloantibodies and distinguish them from benignautoantibodies. iPSC line Relevant Genotype RBC phenotype Antibodydetection 1 Rh null No RHD, inactive D−, C−, E−, c−, e− Identifyantibodies against any high prevalence antigens in Rh RHCE (no Rhantigens) system 2 D−− Inactive RHCE D−, C−, E−, c−, e− Identifyantibodies to RHCE (no RhCE antigens) 3 U−S−s− Inactive GYB D−, U−, S−,s− Identify antibodies against high prevalence U antigen, and againstS/s antigens 4 hrB−, VS+ RHCE*ce(733G) D−, hrB−, VS+ Identify antibodiesagainst high revalence hrB antigen ((−) reaction), or to low prevalenceVS antigen ((+) reaction) 5 hrB−, hrS− RHCE*ce(48C, 667T) D−, hrB−, hrS−Identify specificity antibodies against high prevalence RHCE (hrB vshrS) antigens ((−) reaction) 6 Rh null RHD*DIVa on Rh−null D+, C−, E−,c−, e− Identify antibodies to this antigen which is unique to AfricanGo(a)+ backgroud Go(a)+ Americans 7 Rh−null RHD*DIIIa on Rh−null D+, C−,E−, c−, e− Identify antibodies to this antigen which is unique toAfrican DAK+ backgroud DAK+ Americans 8 Do null Inactive ART D−, Doa−,Dob− Identify antibodies against high prevalence Do and HY antigens.Most useful as a future transfusion product.

TABLE 1.3 Induced pluripotent stem cells (iPSC) generated byreprogramming primary human donor cells or by gene editing. PB,peripheral blood. iPSC lines were generated by reprogramming peripheralblood cells from donors with the exception of the Rh null cell linesthat were generated by CRIPSR/Cas9 gene editing techniques. Cell of RHDRHCE iPSC line Origin genotype Genotype predicted extended antigen typer′S PB DIIIa-CE(4-7)-D ceS Group O, D−, partial C+, E−, partial c+,partial e+, K−, (hrB−, hrS+) DIIIa-CE(4-7)-D ceS Jka+, Jkb−, Fya−, Fyb−,S−, s+, U−, Doa+, Dob + hrs− PB DAU0 ceMO Group O, D+, C−, E−, partialc+, partial e+, K−, Jka+, (hrB^(w+)) DOL ceBI Jkb+, Fya−, Fyb−, S−, s+,U+, Doa−, Dob+ V+VS+ PB RHD ce733G Group O, D+, C−, E−, partial c+,partial e+, K−, Jka+, (hrB−, hrS+) RHD ce733G Jkb−, Fya−, Fyb+, S−, s+,U+, Doa−, Dob + Rh null iPSC Disrupted RHD Group O, D−, C−, E−, c−, e−,K−, Jka−, Jkb+, Fya+, Disrupted RHCE Fyb−, S−, s+, U+, Doa+, Dob+ Lua−b−PB RHD Ce Group O, D+, C+, E−, c+, e+, K−, Jka+, Jkb+, Fya+, Deleted Dce Fyb+, S−, s+, U+, Doa+, Dob+

iPSC Line Method.

The proposed panel of customized iPSCs, listed by priority, is shown inTable 1.2. The first three lines are of highest priority because theywould complement existing routine RBC reagents and allow routinehospital laboratories to rapidly distinguish clinically significantalloantibodies. The remaining lines would allow rapid identification ofthe fine specificity of antibodies and guide selection of compatibledonors. Specific considerations include:

-   -   1. Rh null: In contrast to “Rh negative” RBCs that lack the RhD        antigen only, these Rh null RBCs will lack expression of all Rh        antigens. Few individuals lacking all Rh antigens are reported        worldwide, and there are no Group O living blood donors in the        US so these cells are not available for reagent manufacture.        Individuals with Rh-null RBCs most often have mutations in the        RHAG gene (Rh-associated glycoprotein). RhAG glycoprotein is        required for trafficking of RhD and RhCE to the RBC membrane        (Cherif-Zahar, 1996). However, lack of RhAG is associated with        abnormal RBC morphology, cation defects, and mild anemia. In        contrast, Rh-null RBCs also result when RhD− negative        individuals, who lack RHD, have inactivating mutations in the        RHCE gene. No RBC abnormalities are associated with this Rh-null        genotypic background, and therefore this approach will be used        to engineer Group O, Rh null cells. The Rh null iPSC-derived        RBCs would be a universal reagent for identifying any Rh related        antibody in patient serum, as well as a universal donor cell for        transfusion.    -   The inventors created multiple Rh null iPSC clones from 2 WT        iPSC lines: one WT line that was D+ and one WT line that was D−,        by using CRISPR/Cas9 guide RNAs directed at exon 1 of RHD and/or        RHCE (FIGS. 19A-B). Upon hematopoietic differentiation into        mature iPSC-derived RBCs, flow cytometric analysis for cell        surface Rh antigen demonstrated comparable Rh antigen expression        on on RBCs from donor derived RBCs and an untargeted Rh+ parent        iPSC line, while the targeted Rh null iPSCs produced RBCs        showing no Rh expression on the cell surface (FIG. 19B).    -   2. D −/−: Rare individuals have been described who express D        antigen but lack expression of RhCE (C,c,E,e) antigens due to        inheritance of mutations that cause loss of expression from RHCE        (Reid, 2012). This would be analogous to the “RhD negative”        phenotype which is common (15% of Caucasians), but the “RhCE        negative” phenotype is rare. No morphology changes or anemia is        associated with this phenotype. RBCs engineered with this        phenotype will allow rapid indication that a broadly reactive        antibody is directed specifically to a polymorphism in RhCE        antigens. The availability of these cells for future transfusion        would be broad-ranging and could potentially serve patients with        altered C/c or E/e antigens and parallel the usefulness seen for        “RhD negative” blood usage in patients with no D antigen or        altered D antigen.    -   3. U, S, and s antigen-negative (glycophorin B null): The        absence of glycophorin B, and consequently these antigens on        RBCs has a prevalence of 1% and is exclusive to people of        African Black ethnicity. Hence, patients with SCD are at risk to        make the antibody following routine transfusion. There are no        biological consequences associated with lack of glycophorin B on        RBCs, which is associated with deletion of exon 2, 4 or exon 11        in the GYPB gene. Engineered iPSCs will be generated by        inactivating GYPB in iPSCs that also lack RHD to be most useful        for future transfusion.    -   4 & 5. Negative for high prevalence antigens encoded by RHCE:        These cells would distinguish the fine specificity of antibodies        directed to RHCE proteins, and facilitate improved donor RBC        selection. Table 1.3 shows three lines generated by        reprogramming rare or unusual donor cells, that have        subsequently been differentiated into red blood cells and        express their genotype predicted red cell Rh antigen phenotype.    -   6 & 7. Positive for low prevalence antigens on RhD exclusive to        African Blacks: These cells would express engineered D-epitopes        uniquely found only in African Americans important for        distinguishing fine specificity of antibodies directed to RhD.        The availability of these engineered cells would improve donor        RBC selection by expanding the potential compatible units, as        currently patients who make these antibodies receive RhD        negative blood, which primarily comes from Caucasian donors and        exposes patients to additional antigenic mismatches in other        blood group systems.    -   8. Dombrock (Do) null: RBCs lacking expression of Dombrock        protein, encoded by the ART gene, have been infrequently        described and are associated with several molecular mutations        including skipping of exon 2. The function of this        ADP-ribosyltransferase on RBCs is not known, but rare patients        whose RBCs lack the protein do not have compromised RBC survival        (Gubin, 2000). Antibodies to Doa/b allelic antigens are        notorious for causing severe life-threatening transfusion        reactions (Lomas-Francis, 2010).

The Group O, RhD− lines will be used to generate customized iPSCs withthe exception of the D −/− line, for which the inventors will also usethe Group O, RhD+ lines (Table 1.1). The inventors will design guideRNAs (gRNAs, crispr.mit.edu), screen for efficiency to RHD, RHCE, GYB,and ART, and transfect with Cas9/GFP as described in preliminary data(FIGS. 5A-C). They have initiated this work and successfully identifiedmultiple clones with RHCE disrupted for production of true Rh “null”iPSC lines (FIGS. 19A-B, Table 1.3). Since determination of antibodyspecificity absolutely requires no reactivity to RBCs that lack theoffending antigen, Rh-null iPSC-derived RBCs are essential for antibodyidentification against high prevalence or variant Rh antigens. Combiningtesting using Rh-null and D−− iPSC-derived RBCs, that express D but noRHCE antigens, the anti-RhCE vs anti-D specificities to high prevalenceantigens will readily be distinguished in hospital blood banks.

Reagent iPSC-derived RBCs expressing variant RH antigens will allowidentification of the fine specificity of Rh antibodies. To generatelines that lack the high prevalence RhCE antigens hrB and hrS, theinventors will reprogram rare or uncommon donors, and use the CRISPR/Cas9 technology to generate mutations found in patients: RHCE*ce(733G) andRHCE*ce(48C, 667T) (aka RHce*ceMO) (FIG. 4 ). Simultaneous transienttransfection of Cas9/GFP with gRNAs designed to target a sequence inclose proximity to the desired mutation site, and an ˜200 bpoligonucleotide with the mutant sequence will be used (FIG. 7 ). Theinventors will screen for clones that are homozygous for the mutations.Single nucleotide mutations have been successfully made by introducingmutant oligonucleotides via CRISPR/Cas9 technique into the SETBP1 genewith 7% efficiency for homozygous mutant clones (not shown).Alternatively, they will use homologous directed repair with thestrategy in FIGS. 18A-C.

The true Rh null iPSC lines will be used to generate lines 6 and 7(Table 1.2) to exclusively express the variant RhD antigens Go(a) andDAK. These Rh variants result from multiple mutations in differentexons, and therefore are less amenable to genome editing by CRISPR/Casmethods. Individuals expressing DAK carry a RHD*DIIIa allele thatencodes 5 amino acid changes (FIG. 4 ) and individuals expressing Go(a+)carry a RHD*DIVa allele that encodes 4 amino acid changes. The mutantcDNAs for these RhD variant antigens will be expressed as transgenes inthe “safe harbor” PPP1R12C (AAVS1) locus using the ZFN technology, asdescribed in Preliminary data (FIG. 6 ), or inserted into the endogenousRHCE locus (FIGS. 18A-C). The latter strategy takes advantage of theidentical sequences of exon 1 in both RHD and RHCE alleles. A D− linewas used to knockout RHCE expression via a mutation resulting in anearly stop codon, and the variant RHD or RHCE cDNA can be inserted usinghomology directed repair with a guide targeted to RHCE intron 1. Thesereagent iPSC− derived RBCs will be used to determine the finespecificity of unexplained Rh antibodies that occur in patients despitereceiving “Rh-matched” blood.

Hematopoietic differentiation of the customized iPSCs produced will beperformed according to standard protocols to generate hematopoieticprogenitors (Chou, 2012; Byrska-Bishop, 2015). A 2-step productionprotocol in defined, serum-free media with appropriate combinations ofcytokines will be used to differentiate iPSCs into iPSC-derived RBCs:(1) embryoid body or adherent culture of undifferentiated iPSCs intohematopoietic progenitors, and (2) differentiation of hematopoieticprogenitors into erythroid cells, and their amplification to generateiPSC-derived RBCs. Day 7-9 hematopoietic progenitor cells (HPCs) arepropagated in liquid conditions using SCF, EPO, and holotransferrin toobtain a synchronized population of CD71+ (transferrin receptor) andCD235+ (glycophorin A) iPSC-derived RBCs (FIGS. 8A-B). The matureiPSC-derived RBCs express similar cell surface markers compared to donorRBCs, and are smaller and more morphologically mature (condensed nuclei,mature cytoplasm) than RBCs generated by current protocols fordifferentiation of RBCs from iPSCs. Ongoing work by the inventors aim toestablish culture conditions to generate iPSC-derived RBCs that aredevelopmentally similar to red cells found in the latter part of fetalgestation or postnatally, whereas current protocols generate red cellsfound in a first trimester fetus (FIGS. 10A-C). This is relevant for RBCantigen expression and the goal to have cells from differentdevelopmental stages (embryonic, fetal, adult) since this could provideantigen negative and antigen positive cells.

The HPCs can be cryopreserved and differentiated into iPSC-derived RBCswhen needed. During iPSC-derived RBC differentiation, maximal expansionoccurs by days 12-14 of culture. Myeloid cells may interfere withagglutination studies, so cultures can be sorted to enrich for RBCs thatcomprise 80-85% of the culture. However, the inventors have developedculture conditions such that >95% of cells are mature iPSC-derived RBCs(FIGS. 8A-B), and thus this final enrichment step may not be required ifthe proportion of myeloid cells are consistently low and thus do notinterfere with agglutination testing. Each undifferentiated iPSC givesrise to 2-10 iPSC progenitor cells, which subsequently yields up to 300iPSC− derived RBCs per progenitor cell, such that one 6 well tissueculture plate of undifferentiated iPSCs ultimately produces over 10⁸iPSC-derived RBCs by day 12 of erythroid culture which is sufficient forcharacterization and can undergo scale up for clinical application.

Human ESC-derived RBCs are known to express RhD antigen (Lu, 2008), butexpression of other blood group antigens has not been established. Bloodgroup antigen characterization was performed by flow cytometry, qPCR,and with commercial antibody reagents as well as polyclonal and singlesource sera from NYBC collections (FIG. 25 ). NYBC has a large number ofmonoclonal antibodies to monitor protein expression profiles andepitopes. RBC agglutination of iPSC-derived RBCs with these antibodyreagents will be tested by three common agglutination methods: tube, gelcard and solid phase microplate. Initial studies will be performed onthe primitive, yolk-sac type iPSC-derived RBCs generated with currentprotocols, and subsequently on definitive iPSC-derived RBCs produced.The inventors will also screen for neoantigens which are a concern foriPSC-derived RBCs that may express embryonic, fetal or new proteins thatmight be antigenic. These new antigens would not be detected by testingwith blood group antibodies but may be identified by screening withpools of human sera from donors who might have encountered these andmade natural antibodies.

Since gel card methods require fewer cells than tube methods, the goalwas to optimize iPSC-derived RBCs for this assay. Optimization ofiPSC-derived RBCs for antibody identification was performed first onparental iPSC lines by typing for blood group antigens determined bygenotyping to be expressed on the RBC membrane (Table 1.1). Theinventors demonstrated that only 500 K cells was more than sufficient tovisualize RBCs for macroscopic interpretation of commercial gel cardassays (FIGS. 9A-C). Non-agglutinated RBCs pelleted at the bottom of thecolumn and agglutinated RBCs remained at the top of the column aftercentrifugation.

Using monoclonal Rh typing reagents for D, C, c, E, and e, typing wasperformed with 500,000 iPSC-derived RBCs per gel card assay column.FIGS. 9A-C demonstrate Rh typing for the untargeted D− wild type (WT)iPSC-derived RBCs, the untargeted D+ WT iPSC-derived RBCs, and theCRISPR-targeted Rh null iPSC-derived RBCs. The inventors show that iPSC−derived RBCs agglutinated and remained at the top of the gel matrix whenthe antigen was predicted to be expressed (+) and did not agglutinateand pelleted to bottom of column when antigen was predicted to be absent(−). The Rh null cells did not react with monoclonal antibodies againstthe five principal Rh antigens. In addition to the gel card assay, RBCagglutination will be assessed by performing the tube method and viewedmicroscopically for agglutination, or in 96 well assays in which 1e6cells are incubated at 37° C. with antibody and assessed microscopically(Kim, 2015) or using any of the other exemplary arrays, assays, or kitsdescribed herein.

Since antibodies in patient plasma or serum can have varying titers andwould likely be lower titer than the monoclonal typing reagents, theinventors tested antibody screening with iPSC-derived RBCs againstpatient serum containing known antibodies (FIGS. 21A-B). Two examplesare shown. First, patient serum containing anti-e was reacted with donorderived panel red cells or iPSC-derived RBCs in the gel card assay andshowed agglutination with e+ donor derived RBCs and e+ iPSC-derivedRBCs. Conversely, no agglutination occurred with donor derived e− RBCsor iPSC-derived RBCs, consistent with an anti-e antibody present in thepatient's serum. Second, the inventors tested the iPSC-derived RBCs withpatient serum containing anti-hrS, an antibody to a high prevalenceantigen which is often mistaken as a warm autoantibody due to lack ofappropriate reagent RBCs. Patient serum was reacted with donor derivedpanel red cells or iPSC-derived RBCs in the gel card assay and showedagglutination with all hrS+ cells and no agglutination with all hrS−cells.

Commercial RBC reagents manufactured from donor RBCs are provided assuspensions in buffered preservative solution and have a 4-6 week shelflife. Several storage solutions have been tested for iPSC-derived RBCcompatibility with gel column assays and will be tested for storage.These diluents contain adenine and adenosine to prevent hemolysis andpreserve antigenicity, as well as antibiotics to prevent bacterialcontamination. iPSC− derived RBCs can be suspended at a concentration of200,000 and 400,000 cells/microliter, equivalent to commercial RBCsuspensions respectively, but the inventors will optimize cell numbersfor the different antibody identification techniques used by hospitalblood banks. The inventors will test storage stability and validateperformance of reagent iPSC-derived RBCs. Commercial reagent RBCs areexpected to perform as indicated on the manufacturer package insert andthere is no U.S. standard of potency. They will determine storagestability by maintenance of pH between 5 and 6, measurement of freehemoglobin to assess for hemolysis, and cell morphology assessed bycytospin preparations every 7 days. Maintenance of antigenicity will bemeasured by agglutination studies as described above every 7 days andsuitable performance will be defined as not more than one gradeagglutination difference (i.e., 2+ vs 3+) and with no unexpectedreactivity.

Example 2

Sources of Cells that can be Used to Produce cRBCs and Enucleation.

RBC production starts during the 6^(th) weeks of gestation and continuesthroughout life. Three types of RBCs are successively produced duringdevelopment: primitive RBCs that express embryonic hemoglobins (Hb)(ζ₂ε₂, α₂ε₂, ζ₂γ₂), fetal RBCs that express mostly Hb F (α₂γ₂) and someadult Hbs (α₂β₂, α₂γ₂), and adult RBCs that express mostly adult Hbs andsome Hb F. The precursors of all of these cells can theoretically beused as source of cells but the field has focused on cord blood (CB) andcirculating peripheral blood (PB) hematopoietic stem cells (HSCs), andhematopoietic progenitors cells (HPCs) because they are more readilyavailable. More recently, induced pluripotent stem cells (iPSCs) haveemerged as an additional source of cells to produce cRBCs. CB HSCs/HPCshave been studied extensively because they are more proliferative thanPB cells but there are no available resources from which one couldobtain CB HSCs/HPCs carrying rare blood groups.

One of the first methods to produce cRBCs in liquid culture wasdeveloped by Fibach and colleagues who described a two-step proceduredesigned to first amplify and then favor the maturation of erythroidprogenitors present in umbilical cord and adult peripheral blood (Fibachet al., Blood 73: 100-103, 1989). While this method was effective forthe production of erythroid precursors, it did not yield large amountsof enucleated cRBCs and required serum and conditioned medium.Subsequently a variety of methods were developed that improved on thisprotocol (Freyssinier et al., Br. J. Haematol. 106: 912-922, 1999) andPanzenbock et al., Blood 92: 3658-3668, 1998). The Douay group has madesignificant contributions by developing methods to amplify CB and PBHSCs/HPCs using completely defined conditions (Giarratana et al., Nat.Biotechnol. 23: 69-74, 2005; and Douay et al., Nat. Biotechnol. 23:69-74, 2005). cRBCs produced from CB using these methods express closeto 100% Hb F while those produce from PB HSCs/HPCs express 10-20% Hb Fand 80-90% Hb A.

Other important contributions were made over the last 20 years by theBeug group and others who have developed an erythroblast-expansioncocktail containing stem cell factor (SCF), erythropoietin (EPO), anddexamethasone (SED) to induce extensive proliferation of lateprogenitors and erythroblasts that can differentiate into cellsresembling stress reticulocytes (Leberbauer et al., Blood 105:85-94,2005; Dolznig et al., Methods Mol. Med. 105: 323-344, 2005; and Carottaet al., Blood 104:1873-1880, 2004). cRBCs produced from adultprogenitors using the erythroblast expansion cocktail express 30-60% HbF and 40-70% Hb A.

The present inventors expanded on the studies using hESCs co-cultured ona feeder layer of S17 cells (Kaufman et al., Proc. Natl. Acad. Sci.U.S.A. 98:10716-10721, 2001) by identifying FHB-hTERT as a moreefficient feeder layer and by showing that hESC differentiation towardthe erythroid lineage closely parallels normal human early embryonicdevelopment (Croizat et al., Acta Haematol. 102: 172-179, 1999; Olivieret al., Exp. Hematol. 34: 1635-1642, 2006; Qiu et al., Exp. Hematol.33:1450-1458, 2005; and Qiu et al., Blood 111:2400-2408, 2008). Thelatter point was demonstrated by a detailed analysis of the expressionof the α- and β-like globin genes at the mRNA and protein levels thatrevealed that it was possible to induce hESCs to differentiate intocRBCs containing Hb Gower 1, Hb Gower 2, and Hb F with the same temporalsequence than during normal development.

Several labs have attempted to develop procedures to expand long-termrepopulating HSCs in culture because of their importance fortransplantation applications (Sorrentino, Nat. Rev. Immunol. 4: 878-888,2004). Zhang and colleagues have developed a procedure based on the“STIF” cytokine cocktail (stem cell factor (SCF), thrombopoietin (TPO),insulin-like growth factor 2 (IGF2), and fibroblast growth factor-2(FGF2)) and angiopoietin-like proteins to expand HSCs about 20-fold(Zhang et al., Blood 111:3415-3423, 2008) and Zhang et al., Nat. Med.12: 240-245, 2006). More recently, Boitano and colleagues demonstrated a50-fold HSC expansion using an aryl hydrocarbon receptor antagonist(Boitano et al., Science 329: 1345-1348, 2010).

Maximal expansion of circulating stem and progenitor cells cantheoretically be obtained by successively amplifying the stem cellcompartment, the progenitor compartment, and the erythroblastcompartment (see, e.g., FIGS. 23A-B) (Olivier et al., Stem Cell Transl.Med. 1:604-614, 2012).

Pulsing CD34⁺ cells in HPC-expansion medium for 48 hours prior toincubation in the HSC-expansion medium significantly increased yield. Inbrief, incubation for one week in the HSC-expansion medium increased theyield of cRBCs about 15-fold relative to direct incubation inHPC-expansion medium but pulsing the cells for 48 hours in HPC-expansionmedium prior to incubation in HSC-expansion medium for 5 days increasedthe yield an additional 5- to 20-fold (FIGS. 23C-E).

Longer incubation in HSC-expansion medium (with or without the pulse)resulted in continued increase in the number of CD34⁺38⁻ cells but notin cRBCs yield (Olivier et al., Stem Cell Transl. Med. 1:604-614, 2012),suggesting that the expanding cells were losing erythroid potential. Todetermine if these cells could be redirected toward the erythroidlineage, we introduced periodic 48-hour pulses in HPC-expansion mediumduring long incubation in HSC-expansion medium. After three cycles ofthis regimen (2 days in HPC-expansion, 5 days in HSC-expansion) followedby 1 week in HPC-expansion and 1 week in E-differentiation the overallyield of cRBCs obtained from CB CD34⁺ cells was increased another 15- to20-fold leading to a theoretical overall yield of about 2×10⁷cRBCs/CD34⁺ cell (FIG. 23E).

The mechanisms by which periodic priming with the HPC-expansion mediumincrease the yield of cRBCs in these cultures is not known but severalstudies have shown that in vivo there is a hierarchy of HSCs that aremore or less biased towards the myeloid or lymphoid lineages (Babovic etal., Exp. Cell Res. 329: 185-191, 2014). This suggests that periodicallypriming cells HSC/HPC growing and self-renewing in HSC-expansionconditions might preserve their myeloid potentials and prevent theacquisition of a lymphoid bias. To start testing this hypothesis, HSCs(Lin⁻34⁺38⁻90⁺49f⁺ cells), MPP(Lin⁻34⁺38⁻), andCMP(Lin⁻34⁺38⁺45RA⁻123^(low)) were sorted and cultured in HSC-expansionmedium with or without a pulse. This revealed that pulsing increased thenumber of cRBCs produced by all cell fractions but that the effect ofthe pulse was more pronounced on HSCs and MPPs (data not shown).

As described above, provided herein are novel methods of amplificationof CB HSCs/HPCs, based on combining HSC- and HPC-expansion conditions,that yielded an average of up to 2×10⁷ cRBC/CD34⁺ cells (FIGS. 23A-G).To determine which of the HPC− expansion cytokines (SCF, FLT3-L, IL3,BMP4, IL11, and Epo) are required for the pulse-induced expansion,experiments in which individual and combination of cytokines weresystematically omitted were performed. The inventors showed that cellsproduced in erythroid culture could be frozen and thawed at multipletime point during the procedure with minimal effects on yield. Cellswere frozen at different time points to expedite these experiments.

In addition to measuring the yield of cRBCs, the cultures were monitoredweekly by colony assays to assess the erythroid and myeloid output andby HPLC to assess the type of hemoglobin produced. The 86mmune-phenotypeof the cells in the culture were monitored weekly to determine if cellsgrowing in these conditions for extended period of times hadcharacteristics of HSC, CMP, MEP, or BFUE. The identification wasconfirmed by performing RNA-sequencing experiments and comparing theprofiles with published profiles of hematopoietic stem and progenitorcells.

Because there were no known major surface antigen changes betweenorthochromatic erythroblasts and enucleated cells, it was alsodetermined whether nucleated erythroblasts can be used as reagent redblood cells.

Enucleation.

The first reported method to induce enucleation of culturederythroblasts relied on a feeder layer (Giarratana et al., Nat.Biotechnol. 23: 69-74, 2005). More recently, Miharada et al. (Nat.Biotechnol. 24:1255-1256, 2006) developed culture conditions in whichenucleation occurred without feeder. Both approaches yielded similarenucleation rates (data not shown). Importantly, these results suggestedthat it was possible to obtain close to 100% enucleation with eitherprotocol when PB cells were cultured with the Olivier procedure, butthat the enucleation rate was lower when the erythroblasts expansionprocedure was used or when iPSCs were the source of the cells.

Generation of iPSCs from with Rare Red Cell Phenotypes.

iPSCs with the red blood cell phenotypes identified in Table 1.4 wereproduced. iPSCs were differentiated into CD34⁺ cells using achemically-defined EB formation system that was modified from thespin-EB method (Ng et al., Blood 106:1601-1603, 2005). The same liquidculture methods could generally be used to expand CD34⁺ hematopoieticcells regardless of their sources (Olivier et al., Stem Cell Transl.Med. 1:604-614, 2012). CD34⁺CD38⁻ hematopoietic cells were used as thesource of cells and plasmid nucleofection was used because it wasperformed in GMP conditions using an electroporation buffer that wasmade entirely of inorganic reagents and was easily made under GMPconditions. The iPSCs were grown in E8 medium on vitronectin (Chen etal., Nat. Methods 8:424-429, 2011). All of the components of the culturesystem were commercially available in USP or GMP grade.

The quality of the iPSCs was validated using a protocol which includedfluorescence-activated cell sorting (FACS) analysis for expression ofSSEA-3, SSEA-4, TRA-1-60, and TRA-1-80, and demonstration ofpluripotency by teratoma formation in NSG mice and by embryoid body (EB)formation.

The major difference between PB and iPSC-derived cRBCs was that thelatter cells had an embryonic/fetal phenotype and are a renewal cellsource. Because many antigens were not developmentally regulated, thismight not have preclude the use of iPSCs for the production cRBCs foruse as reagent cRBCs or for transfusion. About 10⁴ cRBCs per PSC-derivedCD34⁺ cells were produced using the Olivier procedure (FIGS. 23C-E) andmore than 1.5×10⁵ CD34⁺ cells could be generated per 6-well plate ofESCs (about 5×10⁶ cells) using a differentiation system based onco-culture with FHB-hTert cells (Olivier et al., Exp. Hematol.34:1635-1642, 2006; Qiu et al., Exp. Hematol. 33L:1450-1458, 2005; andOlivier et al., Stem Cell Transl. Med. 1:604-614, 2012). Using similarPSC-derived CD34⁺ cells, very large amounts of primitive basophilicerythroblasts expressing a mixture of embryonic and fetal Hb weregenerated using the erythroblast expansion procedure (FIG. 23G). Thisresult was confirmed by the existence of erythroblasts that can beexpanded expands nearly indefinitely in the mouse yolk-sac (England etal., Blood 117:2708-2717, 2011). Either the Olivier or theerythroblast-expansion approaches could therefore yield more than 10⁹cRBCs per plate of PSCs.

Alternatively, erythroid progenitors could be immortalized either bytransduction with Sox2, c-Myc, and anti-p53 (Huang et al., Mol. Ther.22:452-463, 2014) or via transduction with Tall and HPV16-E6/E7 cells(Kurita et al., PLoS One 8: e59890, 2013). Both methods immortalizederythroid progenitors in a manner that preserved their ability toterminally differentiate.

Methods to differentiate iPSCs with more adult phenotypes have also beendeveloped (Peraki et al., Mol. Cell Biol. 37(19): e00183-17, 2017).

Generation of O—Rh-null iPSCs. Group O, Rh-null cells, which have beendubbed “universal cells” for transfusion, were useful as reagent RBCsbecause they could be used to rule out the presence of any Rh relatedantibody in the serum of the patient, and could also be useful asuniversal donor cells for transfusion.

The main Rh antigens are D, C, E, c, and e, which are encoded by twoadjacent gene loci, RHD (which codes for the D antigen and whose absenceresults in the Rh negative phenotype) and the RHCE gene (which codes forthe C, E, c, and e antigens). RBCs can be Rh-null because they lackexpression of both the RhD and RhCE, or because they lack expression ofthe RHAG gene, which encodes a protein that forms a complex with theproduct of the RHD and RHCE genes. It has recently been shown that RhAGis important for cation balance in RBCs. RBCs from individuals that lackexpression of RhAG exhibit stomatocytosis and have cation defects.Hence, Rh-null cells that carry deletions of RhAG are not useful asreagents or for transfusion. Patients with deletions of both the RHCEand RHD also have Rh-null red cells, but express RhAG protein in reducedamounts. These are extremely rare and the defect, if any, of the redblood cells has not been studied. Rh-null RBCs, which lacked RhCE andRhD, but had functional RhAG, were generated.

Site-specific mutagenesis in iPSCs was performed using the CRISPR/cas9technology to disrupt expression of the RHCE genes in group O, RhD⁻iPSCs. Donors with this group O RhD negative phenotype were readilyavailable. To disrupt expression of RHCE, guide RNA were designed thattargeted the first or second exon of the RHCE gene and were screened forclones in which repair by non-homologous-end-joining had resulted in theintroduction of a stop codon. iPSCs were first transduced with aninducible Cas9 (e.g., a doxycycline-inducible Cas9) and guide RNAvectors and successfully transduced clones were selected usingantibiotics. Expression of the Cas9 protein was then induced with anagent (e.g., doxycycline) and clones in which mutations were introducedwere screened by PCR.

Maturation and Physical Characterization of cRBCs.

The morphology and maturation of cRBCs was determined by Giemsastaining. Synthesis of Hb was assessed by high performance liquidchromatography (HPLC). The maturation state of cRBCs was also monitoredby measurement of levels of CD235a, CD36, and/or CD71 expression by flowcytometry. Glucose-6-phosphate dehydrogenase (G6PD) and pyruvate kinase(PK) levels were measured as described in (Jansen et al., Am. J.Hematol. 20: 203-215, 1985).

Membrane deformability and protein linkage to the cytoskeleton weremeasured (Dahl et al., Blood 103:1131-1136, 2004; and Dahl et al., Blood101:1194-1199, 2003). These studies included membrane deformabilitymeasured by micropipette aspiration and measurement of the linkage ofintegral proteins to the cytoskeleton by fluorescent imagemicrodeformation (FIMD). G6PD levels and PK levels were similar to thoseseen in young erythrocytes, and that the FIMD experiments providedinsights into membrane and storage stability.

Commercial red cell reagents have a 4-6 week shelf life, however, theactual outdate is 67 days from the day blood is withdrawn from thedonor. Storage stability measurements determined the “outdate” of thecRBC product. Stability was assessed using routine blood bank tests andmaintenance of antigenicity was measured every 5-7 days.

Commercial reagent red cells were provided as 2-4% suspensions inbuffered preservative solution. Two storage solutions were tested. cRBCswere suspended at 2-4% concentration in Immucor manufacturer diluent,which was provided with each set of panel cells received (and availablein excess quantities in the reference laboratory). This diluent solutioncontained adenine and adenosine to retard hemolysis and loss ofantigenicity, and chloramphenicol (0.25 mg/ml), neomycin sulfate (0.1mg/ml), and gentamycin sulfate (0.05 mg/ml) to mitigate bacterialcontamination. Cells were also suspended in Alsever's Solution (Sigma)which permitted storage of RBCs for approximately 10 weeks and wascomposed of an equal volume of 2.05% dextrose, 0.8% sodium citrate,0.055% citric acid, and 0.42% sodium chloride. cRBCs and primary RBCsfrom the donor as control were stored at 1-10° C., with measurement ofhemolysis as free hemoglobin and pH, and the cells were observed formorphology and performance every 5-7 days. Additional RBC storagesolutions and additives, including those found to improve donor RBCstorage (SAGM, MAP) were described in Fung et al., AABB TechnicalManual, Eighteenth Edition, p 138.

Equivalent performance was defined as not more than one grade difference(e.g., 3+ vs. 4+) in agglutination reactions and no unexpectedreactivity. Minimal hemolysis (<5%) was seen at 6 weeks, and pH wasmaintained between pH 5-6. The presence of a bacteriostatic agent wasneeded to prevent bacterial contamination. Storage in dextrose offeredadditional longevity. The pH of storage medium was reported to affectthe rate of antigen deterioration, specifically low pH and low ionicstrength.

Phenotypic and Genotypic Characterization of cRBCs.

The expression of clinically relevant RBC antigens was determined bygenetic polymorphisms in genes that were often regulated duringdifferentiation. Comparison of blood group antigen expression on cRBCscompared to primary RBCs from rare blood donors provided indications onwhich cell sources were appropriate for the reagent cRBCs applicationand for transfusion. Enucleation rates in culture varied significantlybetween sources of cells. Culture methods were one of the majordeterminants of overall cell yield. Antigen profiles of enucleated cRBCswere compared with that of basophilic and orthochromatic erythroblaststo determine if red cell precursors could be used as reagent RBCs. Bloodgroup antigens were encoded throughout the human genome and manyantigens were present in protein complexes.

The cRBCs produced as described above in Example 2 were compared withnative cells from the donors which were stored frozen in liquid nitrogenby routinely used methods. Experiments are performed with enucleatedcells and with precursors. FIG. 24 summarized the serologic andmolecular FDA-licensed methods and techniques that were used tocharacterize the cRBCs, as well as laboratory developed tests (LDTs)using serologic reagents and molecular methods including genesequencing.

Serologic Typing for Blood Group Antigens.

Testing was performed by standard tube methods with commercial reagentsand polyclonal and single source sera from our collections. Supernatantfluids containing monoclonal antibodies that were specific for the bloodgroups in Table 2 were also used.

TABLE 2 Blood Group Systems for Testing Genetic Changes chromosomal.System name # of antigens Gene names location ABO 4 ABO 9q34.2 MNS 46GYPA, GYPB 4q31.21 Rh 52 RHD, RHCE 1p36.11 Lutheran 20 LU, BCAM 19q13.32Kell 34 KEL 7q34 Duffy 5 FY, DARC 1q23.2 Kidd 3 JK, SLC14A1 18q12.3Diego 22 DI, SLC4A1 17q21.31 Yt 2 YT, ACHE 7q22.1 Scianna 7 SC, ERMAP1p34.2 Dombrock 8 DO, ART4 12p12.3 Colton 4 CO, AQP1 7p14.3Landsteiner-Wiener 3 LW, ICAM4 19p13.2 RhAG 4 RHAG 6p12.3 JR 1 JR, ABCG24q22.1 LAN 1 LAN, ABCB6 2q36

Genotyping for Blood Group Antigens.

In addition to serologic typing, the antigen type of the original RBCsand cRBCs were tested by genotyping which also provided information onchromosomal loss or genetic changes. Genotyping was also required forantigen specificities that were difficult or impossible to characterizeby serology alone. DNA was extracted and human erythrocyte antigen (HEA)genotyping for 35 antigens in 12 different systems, as well as RHDgenotyping of the original sample and the recombinant cRBCs, wasperformed. High resolution RH genotyping for altered Rh proteins wasperformed as described in Jansen et al., Transfusion 53(4): 741-746,2012. Genotyping was monitored for chromosomal loss in culture across alarge number of blood group loci.

FACS and Western Blot Detection of Antigen, Glycosylation, and ProteinMembrane Structural Changes.

Flow cytometry and quantitative Western blot was performed to moreprecisely monitor antigen expression levels of glycophorin A-GPA (MN),glycophorin B-GPB (SsU), Band 3 (Diego), Rh, RhAG, GLUT1, Kell, Duffy,CD59, CD47, CD71, and Colton AQP1 (aquaporin-1). Changes in antigen,glycosylation, and protein expression profiles and epitopes wasmonitored and mapped. These techniques were used when the standardtyping methods provided clues (over or under expression, abnormalreactivity) that new antigens might be present on cRBCs.

Poly-Agglutination.

RBCs were monitored for poly-agglutination associated with alteration ofcarbohydrate modifications. Normal human sera had naturally occurringantibodies to carbohydrate epitopes associated with the common bloodgroup antigens (anti-A, anti-B) but also had antibodies to othercarbohydrate containing structures that occurred in nature (anti-T,anti-Tn). Pools of normal human sera were incubated with the cRBCs andwere observed for agglutination by macro- and microscopic methods.

Compatibility Testing.

cRBCs were tested for cross-match compatibility in the same manner asoriginal cells against normal human serum and sera from patients withauto- and allo-antibodies with known specificity.

Screening for Neo Antigens.

A concern with cRBCs, particularly those produced from iPSCs, was thatthey might express fetal or abnormal proteins or epitopes that might beantigenic. These neo-antigens were unlikely to be detected by testingwith blood group antibodies, but might be detected by screening with alarge number of human sera from patients and donors who might haveencountered these neo-antigens and made natural antibodies.

These important experiments allowed us to determine if cRBCs anderythroid precursors produced by extensive expansion of PB andiPSC-derived CD34⁺ expressed the same antigen profiles as RBCs producedin vivo.

Minor changes were detected by studying mRNA expression byRNA-sequencing or microarray analysis. Exposure of neo-epitopes on cRBCsalso revealed by testing with antibodies to structural components of theunderlying RBC cytoskeleton not normally exposed at the cell surface(e.g., spectrin, ankyrin, 4.1, and 4.2).

All of the compositions and/or methods disclosed and claimed herein canbe made and executed without undue experimentation in light of thepresent disclosure. While the compositions and methods of thisdisclosure have been described in terms of embodiments, it will beapparent to those of skill in the art that variations may be applied tothe compositions and/or methods and in the steps or in the sequence ofsteps of the method described herein without departing from the concept,spirit and scope of the disclosure. More specifically, it will beapparent that certain agents which are both chemically andphysiologically related may be substituted for the agents describedherein while the same or similar results would be achieved. All suchsimilar substitutes and modifications apparent to those skilled in theart are deemed to be within the spirit, scope and concept of thedisclosure as defined by the appended claims.

It is to be understood that while the invention has been described inconjunction with the detailed description thereof, the foregoingdescription is intended to illustrate and not limit the scope of theinvention, which is defined by the scope of the appended claims. Otheraspects, advantages, and modifications are within the scope of thefollowing claims.

VIII. REFERENCES

The following references, to the extent that they provide exemplaryprocedural or other details supplementary to those set forth herein, arespecifically incorporated herein by reference.

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What is claimed:
 1. A plurality of antigenically distinct engineered redblood cells (RBCs), wherein said plurality of RBCs exhibit distinctblood antigen group profiles, including at least two rare blood antigengroups, wherein: (a) said plurality of RBCs are produced from inducedpluripotent stem cells using CRISPR to insert, delete or disrupt acoding sequence for one or more blood antigens; (b) said plurality ofRBCs comprise two or more of the following blood antigen group profiles:Rh null, D −/−, U/S/s antigen negative (glycophorin B null), RHCEnegative, positive for low prevalence RhD antigens, Dombrok (Do) null,and Lutheran null; or (c) said plurality of RBCs further comprise one ormore of Kell positive, Kidd positive, Duffy positive and MNS antigenpositive.
 2. The plurality of antigenically distinct engineered RBCs ofclaim 1, wherein said plurality of RBCs exhibit at least three, four,five, six, seven, eight, nine, ten or fifteen distinct blood antigengroups.
 3. The plurality of antigenically distinct engineered RBCs ofclaim 1, wherein said plurality of RBCs are immortalized fromnaturally-occurring isolated RBCs.
 4. The plurality of antigenicallydistinct engineered RBCs of claim 3, wherein said plurality of RBCs areimmortalized by transfecting a naturally-occurring RBC with a constructexpressing a transforming oncoprotein.
 5. The plurality of antigenicallydistinct engineered RBCs of claim 1, wherein said plurality of RBCs areproduced from induced pluripotent stem cells.
 6. The plurality ofantigenically distinct engineered RBCs of claim 1, wherein saidplurality of RBCs are produced from induced pluripotent stem cells usingCRISPR to insert, delete or disrupt a coding sequence for one or moreblood antigens.
 7. The plurality of antigenically distinct engineeredRBCs of claim 1, wherein said plurality of RBCs comprise two or more ofthe following blood antigen group profiles: Rh null, D −/−, U/S/santigen negative (glycophorin B null), RHCE negative, positive for lowprevalence RhD antigens, Dombrok (Do) null, and Lutheran null.
 8. Theplurality of antigenically distinct engineered RBCs of claim 6, whereinsaid plurality of RBCs further comprise one or more of Kell positive,Kidd positive, Duffy positive and MNS antigen positive.
 9. The pluralityof antigenically distinct engineered RBCs of claim 1, wherein saidplurality of RBCs comprise three, four, five or all six blood antigengroup profiles.
 10. A recombinant red blood cell, wherein therecombinant red blood cell is characterized by the absence of at leastone or more cell surface antigens on its surface selected from the groupconsisting of: a C antigen, an E antigen, a c antigen, an e antigen, a Uantigen, an S antigen, an s antigen, an hrB antigen, a Lua antigen, aLub antigen and a CD47 antigen, and wherein: (a) the recombinant redblood cell is characterized by the absence of at least ten of the one ormore cell surface antigens; or (b) the recombinant red blood cell ischaracterized by the absence of at least eight of the one or more cellsurface antigens; or (c) the recombinant red blood cell is characterizedby the absence of at least four of the one or more cell surfaceantigens; or (d) the recombinant red blood cell is characterized by theabsence of at least two of the one or more cell surface antigens; or (e)the recombinant red blood cell is further characterized by the absenceof a D antigen on its cell surface; or (f) wherein the recombinant redblood cell is further characterized by the presence of a D antigen onits cell surface; or (g) the recombinant red blood cell is furthercharacterized by the presence of a Go(a) antigen on its cell surface; or(h) the recombinant red blood cell is further characterized by thepresence of a DAK antigen on its cell surface; or (i) the recombinantred blood cell is characterized by the absence of a Doa antigen and aDob antigen on its cell surface.
 11. The recombinant red blood cell ofclaim 10, wherein the recombinant red blood cell is characterized by theabsence of at least ten of the one or more cell surface antigens. 12.The recombinant red blood cell of claim 10, wherein the recombinant redblood cell is characterized by the absence of at least eight of the oneor more cell surface antigens.
 13. The recombinant red blood cell ofclaim 10, wherein the recombinant red blood cell is characterized by theabsence of at least four of the one or more cell surface antigens. 14.The recombinant red blood cell of claim 10, wherein the recombinant redblood cell is characterized by the absence of at least two of the one ormore cell surface antigens.
 15. The recombinant red blood cell of claim10, wherein the recombinant red blood cell is further characterized bythe absence of a D antigen on its cell surface.
 16. The recombinant redblood cell of claim 10, wherein the recombinant red blood cell isfurther characterized by the presence of a D antigen on its cellsurface.
 17. The recombinant red blood cell of claim 10, wherein therecombinant red blood cell is further characterized by the presence of aGo(a) antigen on its cell surface.
 18. The recombinant red blood cell ofclaim 10, wherein the recombinant red blood cell is furthercharacterized by the presence of a DAK antigen on its cell surface. 19.The recombinant red blood cell of claim 10, wherein the recombinant redblood cell is characterized by the absence of a Doa antigen and a Dobantigen on its cell surface.
 20. A kit comprising: a solid support; afirst reagent red blood cell characterized by the presence of one ormore cell surface antigens on its surface; and a second reagent redblood cell characterized by the absence of at least one of the one ormore cell surface antigens on its surface; wherein: the one or more cellsurface antigens are selected from the group consisting of: a C antigen,an E antigen, a c antigen, an e antigen, a U antigen, an S antigen, an santigen, an hrB antigen, a Lua antigen, a Lub antigen and a CD47antigen, and wherein: (a) the recombinant red blood cell ischaracterized by the absence of at least ten of the one or more cellsurface antigens; or (b) the recombinant red blood cell is characterizedby the absence of at least eight of the one or more cell surfaceantigens; or (c) the recombinant red blood cell is characterized by theabsence of at least four of the one or more cell surface antigens; or(d) the recombinant red blood cell is characterized by the absence of atleast two of the one or more cell surface antigens; or (e) therecombinant red blood cell is further characterized by the absence of aD antigen on its cell surface; or (f) wherein the recombinant red bloodcell is further characterized by the presence of a D antigen on its cellsurface; or (g) the recombinant red blood cell is further characterizedby the presence of a Go(a) antigen on its cell surface; or (h) therecombinant red blood cell is further characterized by the presence of aDAK antigen on its cell surface; or (i) the recombinant red blood cellis characterized by the absence of a Doa antigen and a Dob antigen onits cell surface; and (j) the first reagent red blood cell and thesecond reagent red blood cell have the same surface phenotype, exceptfor the at least one cell surface antigen.